FS1 Unit 3 and 4 Handouts FA24.pdf

Foundational Sciences I Table of Contents Unit 3 Session 25. Protein Synthesis (Werning)...................................................................................................... 2...

Foundational Sciences I Table of Contents Unit 3 Session 25. Protein Synthesis (Werning)...................................................................................................... 2 Session 26-27. Translation & Protein Processing I and II (Ananieva)......................................................... 13 Session 28. Lipids, Mitochondria & Storage (Werning).............................................................................. 33 Session 29. Digestion, Recycling & Waste Disposal (Werning)................................................................... 42 Session 30. Intro to Metabolism (Wilson).................................................................................................. 51 Session 31. Carbohydrate Diges on & Uptake (Schmidt).......................................................................... 63 Session 32. Glycolysis (Schmidt)................................................................................................................. 75 Session 33. Acetyl CoA & the Citric Acid Cycle (Wilson)............................................................................. 88 Session 34. Mitochondrial Electron Transport & ATP Generation (Wilson)............................................... 99 Session 35. Gluconeogenesis & Pentose Phosphate Pathway (Schmidt)................................................. 109 Session 36. Glycogen Metabolism, Ethanol Metabolism (Schmidt)......................................................... 117 Session 37. Extracellular Matrix, Glucuronida on, Organ Specific Carbohydrate Metabolism (Schmidt)..................................................... 127 Unit 4 Session 38. Extracellular Matrix (Werning)............................................................................................... 135 Session 39. Lipid Digestion, Uptake & Oxidation (Schmidt)..................................................................... 146 Session 40. Lipid Synthesis & Regulation of Lipid Metabolism (Schmidt)................................................ 158 Session 41. Lipid Transport & Presentation of Dyslipidemias (Schmidt).................................................. 169 Session 42. Cholesterol & Phospholipids (Schmidt)................................................................................. 179 Session 43. Plasmalogens, Sphingolipids & Lipid Signaling (Schmidt)...................................................... 189 Session 44.-45. Protein & Amino Acid Metabolism I - II(Ananieva).......................................................... 199 Session 46.-47. Protein & Amino Acid Metabolism III - IV (Ananieva)...................................................... 222 Session 48. Protein & Amino Acid Metabolism V (Wilson)....................................................................... 250 Session 49. Nutritional Status (Schmidt)................................................................................................... 260 Session 50. Biochemistry of Diabetes Mellitus (Schmidt)......................................................................... 271  FS1 25 / MSK 08 – Protein Synthesis – Fall 2024 Dr. Sarah Werning – [email protected] Lab 05 is associated with this lecture Recommended Readings GTH – Ch 2 (Cytoplasm): Protein Synthetic and Packaging Machinery of the Cell (Skip Fig 2.13) + Figs 2.5, 2.6; Ch 3 (Nucleus): Figs 3.12, 3.13 only; Ch 9 (Nervous Tissue): Neuronal Cell Body (Soma, Perikaryon) only (stop at Inclusions) GCA – Ch 1 (The Cell): Ribosomes; Endoplasmic Reticulum; Golgi Apparatus, cis- Golgi Network, and trans-Golgi Network; Histophysiology: II. Protein Synthesis and Exocytosis; Graphic 1-4 (Protein Synthesis and Exocytosis); Plates 1-1 (Typical Cell), 1- 2 (Cell Organelles and Inclusions; focus on Figs 3 & 4), 1-5 (Typical Cell, Electron Microscopy), 1-8 (Golgi Apparatus, Electron Microscopy) o All graphics and plates listed above are helpful for Lab 5 W – Ch 1 (Cell Structure and Function): Protein Synthesis, Figs 1.6, 1.7, 1.9, 1.10, 1.15, 1.20a; Ch 14 (Gastrointestinal tract): Fig 14.23 only; Ch 15 (Liver and Pancreas): Fig 15.15c only; Ch 17 (Endocrine System): Fig 17.3d only o All figures listed above are helpful for Lab 5 BRS – Ch 1 (Cell): sections VII.A.1,2,6 (Organelles: Ribosomes, RER, Golgi apparatus; Skip Table 1.3), VIII.B (Protein Synthesis), XVI (Ribonucleic Acid); Fig 1.26, 1.27 --------------------------------------------------------------------------------------------------------------------- Lecture Objectives See last 2 pages for example open-ended study questions for each lecture objective. After reviewing this lecture, the learner should be able to: 1. Relate the structure of the organelles involved in protein synthesis to their functions. 1a. nucleolus 1b. small & large ribosomal subunits, ribosomes, & polyribosomes 1c. RER (rough endoplasmic reticulum) 1d. Golgi apparatus 1f. transport & secretory vesicles 2. Describe how ribosomal subunits are synthesized and how they assemble into ribosomes and polyribosomes. 3. Compare & contrast how proteins are synthesized in the cytosol vs. the rough endoplasmic reticulum. Lecture Outline I) Relevant Nucleic Acids A) DNA human genome = nuclear genome + mitochondrial genome nuclear genome is composed of nuclear DNA (nucDNA) – 23 pairs of chromosomes nuclear genome can be divided into 2 parts: ‒ coding regions (protein-coding regions): genes that code for polypeptides/proteins ‒ very little of the human nuclear genome encodes proteins (~1.5%) ‒ “codes for”, “encodes” = specifies the amino acid sequence for a polypeptide/protein the cell will synthesize ‒ noncoding DNA = DNA sequences that do not code for proteins ‒ most noncoding DNA is actually functional! o instructions for making tRNA, rRNA, regulatory DNA/RNA, & structural components ‒ some noncoding DNA is nonfunctional, but more functions are discovered every year  not “junk DNA” B) RNA mRNA (messenger RNA) ‒ coding RNA; the template for protein synthesis ‒ transcribed from the coding regions of DNA (unlike tRNA & rRNA) ‒ contains the recipe for making a protein tRNA (transfer RNA) ‒ adapter molecule; binds amino acids & carries them to the correct sites on mRNA during protein synthesis ‒ one end is the anticodon (complement sequence to an mRNA codon) ‒ the other end is attached to an amino acid rRNA (ribosomal RNA) ‒ combined with proteins to form the 2 ribosomal subunits C) Main Processes Involving Nucleic Acids: ‒ replication: exact DNA copy of the genome is made prior to mitosis ‒ not really part of protein synthesis, but can introduce errors ‒ transcription: a DNA gene is copied (rewritten) into a new molecule of RNA ‒ in terms of protein synthesis, usually refers to DNA  mRNA ‒ translation: ribosomes synthesize a protein based on the mRNA instructions ‒ errors in any of these processes can lead to dysfunctional proteins II) Organelles Involved in Protein Synthesis A) Ribosomal Subunits (2) small and large ribosomal subunits (SRU, 40S subunit & LRU, 60S subunit) ‒ subunits unite to form a ribosome at the start of protein translation (+ more later)  ‒ composition: rRNA & ribosomal proteins ‒ location: assembled in nucleolus, exit nucleus via nuclear pores ‒ imaging: individual ribosomal subunits cannot be resolved in TEM or LM B) Ribosomes & Polyribosomes ribosomes – coordinate & synchronize alignment of tRNA & mRNA to assemble polypeptides/proteins from amino acids, in a sequence specified by mRNA ‒ composition & structure: large + small ribosomal subunits; no membrane ‒ form at the start of protein synthesis, when a large & small subunit unite ‒ disassociate back into subunits once the polypeptide chain is synthesized ‒ location: free in the cytosol (unattached to a membrane), or attached to the cytoplasmic surfaces of the RER or outer nuclear membrane ‒ imaging: individual ribosomes are resolvable in TEM, but not LM ‒ TEM: distinct, round, & electron-dense; may be attached to RER or outer nuclear membrane, or floating in cytosol ‒ LM: H&E – contributes to basophilia of the cytoplasm ⇒ if cytoplasm stains intensely basophilic, you can infer that the cell is actively synthesizing protein, usually for secretion polyribosomes (polysomes) – several ribosomes bound to the same mRNA template, like beads on a string  allows many copies of a protein to be synthesized from the same mRNA template in a short amount of time ‒ greatly improves efficiency of translation vs. isolated ribosomes ‒ location: form wherever ribosomes form (cytosol, RER, outer nuclear membrane) ‒ free polyribosomes in cytosol  not attached to a membrane ‒ imaging: resolvable in TEM, but not LM ‒ TEM: close clusters of ribosomes; in the cytosol, they often form a spiral ‒ LM: H&E – contributes to the basophilia of the cytoplasm C) Cytosol one of several places where ribosomes & polyribosomes translate mRNA  protein imaging: resolvable in TEM, but not LM ‒ TEM: look for ribosomes & polyribosomes in the spaces between organelles ‒ LM: H&E – cytoplasm is basophilic when lots of protein is being synthesized for secretion (from RER + free polyribosomes) ‒ in cells that do regulated (signal-dependent) secretion: basophilic cytoplasm at one end + clusters of acidophilic secretory granules D) Rough Endoplasmic Reticulum (RER) one of several places where ribosomes & polyribosomes translate mRNA  protein also sequesters proteins as they are translated, performs initial post-translational modification of certain types of proteins & monitors protein quality  composition & structure: ‒ complex & continuous network of cisterns fluid-filled, membrane-bound sacs & tubules whose cytoplasmic surfaces are studded with ribosomes & polyribosomes RER membrane is continuous throughout the RER (all cisterns connected) ‒ also continuous with membrane of SER & outer nuclear membrane RER lumen is continuous throughout the RER (all cisterns connected) ‒ also continuous with SER cisterns & with perinuclear space location: extends throughout much of the cytoplasm at least some portion is always nucleus-adjacent also associated with Golgi imaging: individual cisterns (lumen, membrane, ribos) resolvable in TEM, but not LM TEM: “flattened sacs in large parallel stacks”; long & thin, and studded with ribosomes & polyribosomes o ribosomes make the RER surface look “rough” vs SER o extensive in cells whose major function is protein secretion  can occupy half of the cytoplasm (or more) LM: H&E – contributes to the basophilia of the cytoplasm E) Golgi apparatus (Golgi complex/body) protein sorting & distribution centers modify proteins produced in the RER, sort them, & package them into vesicles for secretion or intracellular transport composition & structure: each Golgi consists of several (3-10) slightly curved & parallel cisterns (fluid- filled, membrane-bound sacs) ‒ not as many cisterns as RER & they are confined to a smaller area ‒ cisterns are flattened with dilated edges, & lack ribosomes ⇒ vesicles commonly fusing to or budding off dilated edges ‒ usually more than 1 Golgi per cell cisterns are attached to microtubules  facilitates fast transport of vesicles has a definite orientation, each part does different types of protein modification ‒ cis Golgi network (CGN): cisterns on the same side as RER ‒ medial Golgi cisterns: middle cisterns ‒ trans Golgi network (TGN): cisterns on the opposite side vs RER location: close to RER imaging: resolvable in TEM & sometimes inferred in LM (Golgi ghost) TEM: an entire Golgi often resembles a rainbow o cis-Golgi is the “top of the rainbow” or outside curve o trans-Golgi is the “bottom of the rainbow” or inside curve  o individual cisterns look like pita pockets, with lumens narrower in the center & dilated edges o common to see vesicles fusing to or budding off dilated edges, or clustered near the trans-Golgi LM: Golgi unstained in H&E, but may show a negative staining patten o H&E – if cytoplasm is basophilic, you may see a Golgi ghost  pale region near the nucleus; unstained Golgi stands out against the dark, basophilic background o IHC – antibodies against Golgi-specific proteins F) Vesicles transport vesicles & secretory vesicles (secretory granules) composition, structure, & location: varies depending on content imaging: always resolvable in TEM; sometimes resolvable in LM TEM: vary in size; membranes are electron-dense, but contents may be electron-lucent or electron-dense o transport vesicles are small & lie between RER & cis-Golgi, or close to the edges of the cis- & medial cisterns o storage vesicles are larger & close to the trans-Golgi o secretory vesicles may accumulate near the cell membrane LM: H&E – most visible in protein-secreting cells as acidophilic granules concentrated in one end of the cell III) Protein Synthesis A) RNA Synthesis & Assembly of the 2 Ribosomal Subunits this part is the same for all proteins encoded by nuclear DNA!!! mRNA & tRNA are transcribed in the nucleoplasm mRNA & tRNA exit the nucleus through nuclear pores  cytoplasm small & large ribosomal subunits (SRU & LRU): noncoding DNA is transcribed into rRNA in the nucleolus ribosomal proteins are synthesized in the cytosol & transported to nucleolus both subunits are assembled in the nucleolus  process governed by nucleolin subunits are modified (methylated) in the nucleolus B) Where Protein Synthesis Occurs subunits leave the nucleus as separate entities via nuclear pores  not ribosomes yet! subunits travel to one of several location surface of outer nuclear membrane, mitochondria, cytosol, or surface of RER location of protein synthesis is related to where that protein will be sent/used  DR WERNING’S AWESOME PROTEIN SYNTHESIS LOCATION CHEAT SHEET Ribosomes (or polyribosomes) translate mRNA into proteins, regardless of their location! Ribosome location Proteins synthesized Where those proteins will be sent integral membrane surface of the outer proteins for the inner & outer nuclear membranes nuclear membrane nuclear envelope proteins specific to mitochondria stay in the mitochondrion or its membranes mitochondria cytoskeletal proteins peripheral membrane none of these proteins stay in the cytosol are toxic nor physically proteins used in the destructive to cytosol cytosol the cell proteins used in the nucleus via membrane nucleoplasm & these are not transport (incl. nuclear pores) nucleolus transported in vesicles proteins used in most organelles via membrane organelles’ lumens transport outside the cell proteins that will be all of these (many are toxic or physically secreted are kept in destructive to the cell) the RER, lysosomes & late endosomes Golgi, or a lysosomal proteins (these can all destroy a cell) vesicle from surface of the RER the moment integral membrane they are proteins for the cell inserted into a membrane synthesized, membrane & (many are receptors that can to protect cytoplasmic organelle bind cytoplasmic structures) the cell! membranes C) Subunit Functions During Translation SRU ensures correct pairing between mRNA codon & tRNA anticodon 1 binding site for mRNA & 3 sites for tRNA binding does not happen until the SRU has exited the nucleus LRU contains enzymes that catalyze the formation of peptide bonds D) Proteins Synthesized by Free Polyribosomes in the Cytosol all of these proteins will be used inside the cell (see chart on previous page) Ribosome & Polyribosome Assembly in the cytoplasm, an SRU binds an mRNA & activated tRNAs codons on mRNA base-pair with anticodons on tRNA a tRNA recognizes a start codon on the mRNA LRU binds to the complex  now it is officially a ribosome o LRU contains an enzyme that catalyzes peptide bond formation between amino acids, so that they can be added to the growing polypeptide chain  translation has now begun as the mRNA strand feeds through the ribosome, other ribosomes begin to form on the portions that have already translated by the first ribosome  a free polyribosome has now formed explanation: ribosomes translate from start to finish; can’t start in the middle ‒ only working with a small amount of the mRNA strand at once ‒ as they translate, the mRNA feeds out behind them ‒ then, other ribosomes latch on to the same mRNA strand & start reading it ⇒ a single strand of mRNA can have ~15 ribosomes attached to it Translation & Post-Translational Modification translation continues until a stop codon is reached stop codon causes the ribosome to release the polypeptide chain  translation is now complete ribosomal subunits detach from each other  ribosome has disassembled o subunits stay in the cytosol o they can be reused to translate any mRNA strand post-translational modification of these proteins occurs within the cytosol ‒ the newly-formed protein is in linear form (1° structure); it must be folded (and otherwise modified) before it can be functional ‒ folded by chaperonins (chaperone proteins) in the cytosol E) Proteins Synthesized by Polyribosomes on the RER Surface all of these proteins require packaging in vesicles (see chart) these proteins are segregated into the lumen of the RER cisterns during translation & transferred via membrane-bound vesicles at all stages afterward i.e., they will never be allowed to contact the cytosol! Ribosome & Polyribosome Assembly & Initial Translation in the cytoplasm, an SRU binds to an mRNA  ‒ mRNA has a signal sequence at the beginning (5’ end) that indicates it needs to be translated at the RER surface activated tRNAs bind to the SRU codons on mRNA base-pair with anticodons on tRNA a tRNA recognizes a start codon on the mRNA LRU binds to the complex  now it is officially a ribosome o LRU contains an enzyme that catalyzes peptide bond formation between amino acids, so that they can be added to the growing polypeptide chain  translation has now begun as the mRNA strand feeds through the ribosome, other ribosomes begin to form on the portions already translated by the first ribosome  a polyribosome has now formed Translocation of Polypeptides into the RER Lumen while the polypeptide chain is being translated on its surface, the RER moves the end of the growing chain into its lumen, sequestering it from the cytosol first, the signal sequence is translated into the signal protein a signal recognition particle (SRP) recognizes the completed signal protein & binds it, temporarily pausing translation (stops adding polypeptides to chain) SRP binds a receptor on the RER’s cytosolic surface, stimulating these events: o a pore opens in the RER membrane o SRP is released from the signal protein o the developing polypeptide chain is fed through the pore into the lumen of the RER cistern  this is called co-translational translocation ⇒ because the beginning of the polypeptide chain is being relocated into the RER lumen *at the same time* that the middle and end of the sequence are being translated enzymes in the cistern lumen cleave the signal protein from the chain o this “unpauses” the translation process as the remainder of the polypeptide chain is assembled, it is fed directly into the lumen of the RER cisternal o chaperone proteins (chaperonins) in the RER lumen assist in “pulling” the polypeptide chain into the lumen a stop codon is reached, causes the polypeptide chain to release the ribosome  translation is now complete ribosomal subunits detach from each other  ribosome has disassembled ‒ subunits stay in the cytosol; can be reused to translate any mRNA strand  Initial Modification by Chaperonins in the RER after the polypeptide chain has been completely moved into the RER lumen, chaperonins in the lumen do several initial modifications to that new protein: o initial (core) glycosylation  attach an oligosaccharide side chain o linear proteins are folded into their globular (3D) form ⇒ proper folding is guided by chaperone proteins & RER enzymes ⇒ protein is folded around the core oligosaccharide o assembly of multichain proteins RER also monitors protein quality & inhibits protein aggregation The rest of this description assumes everything has gone smoothly, yielding a perfectly folded but immature protein. We will discuss what happens when proteins are malformed in the next lecture. Transfer to the cis-Golgi Network (CGN) newly-formed proteins are sent from the RER to the CGN via transport vesicles, which bud off the RER’s membrane o reminder: these are coatomer-coated vesicles o vesicles are transferred along microtubules via motor proteins vesicles dock with the CGN membrane & merge  proteins have now been transferred to lumen of Golgi cisterns Golgi Modification & Sorting of Proteins further modifies the proteins assembled in the RER ‒ mostly, these modifications involve adding things to proteins, e.g.: ⇒ chemical shipping labels ⇒ additional glycosylation ⇒ sulfation ‒ proteins are modified in the CGN, then transferred to medial cisterns ‒ proteins further modified in medial cisterns, then transferred to the TGN ‒ transfers via transport vesicles going between Golgi cisterns sorts proteins by destination, based on their chemical “shipping labels” ‒ “shipping labels” = signaling molecules indicate its ultimate destination within or outside the cell ⇒ labels indicate the cargo is coming from Golgi & going to a specific location (see chart on page 6) packages proteins into vesicles & ships them to final destination Post-translational modification of proteins by RER & Golgi is covered in detail in other lectures Example Open-Ended Study Questions Study questions illustrate how each objective might be studied at appropriate detail for the exam. 1. Relate the structure of the organelles involved in protein synthesis to their functions. 1a. How would you distinguish the RER from the Golgi in a TEM image? Things to consider: - What features of the RER membrane reflect its role in protein synthesis? - Why are the edges of Golgi cisterns dilated? - What is the position of each organelle, relative to each other and to the nucleus? - Which one is more likely to have numerous vesicles in close association, and why? 1b. Describe the H&E staining patterns of a cell actively synthesizing large amounts of protein for secretion via exocytosis (include info on cytoplasm, RER, Golgi, vesicles, & nucleus). - Which of these structures would be most prominent in a TEM image of the same cell? - Would LM staining patterns differ if the proteins being synthesized were destined to be used in the cell’s cytoskeleton or cytosol, rather than secreted? 1c. How do polyribosomes improve protein translation efficiency compared to solo ribosomes? 1d. Which structures discussed in this lecture are electron-dense? Which are electron-lucent? Why do vesicles vary in electron density? 1e. What is a “Golgi ghost”? When and why does it occur in H&E staining? 1f. How do the cis-Golgi network, medial cisterns, and trans-Golgi network differ in terms of their functions and location? How would you distinguish them in TEM images? 2. Describe how ribosomal subunits are synthesized and how they assemble into ribosomes and polyribosomes. 2a. What is the difference between ribosomal subunits, ribosomes, and polyribosomes? Where are each assembled? What is the function of each? What happens to each after protein synthesis is complete? 2b. Where are mRNA, tRNA, and rRNA synthesized? Which of these is associated with the protein nucleolin during synthesis? What is the role of each in protein translation? 2c. How does the small ribosomal subunit differ in function from the large ribosomal subunit? 3. Compare & contrast how proteins are synthesized in the cytosol vs. the RER. 3a. How does the sequence of mRNA coding for proteins that will be synthesized in the cytosol differ from mRNA that codes for proteins synthesized at the RER surface? 3b. Explain how a ribosome becomes “docked” to the cytosolic surface of the RER. What events does docking stimulate? 3c. What does the phrase “co-translational translocation” mean? When & where does this occur? 3d. Which proteins are synthesized in the cytosol, and which are synthesized at the RER surface? Where are each folded and modified? How are each moved through the cytoplasm? 3e. How do the post-translational modifications of the RER differ from the those of the Golgi? 3f. List two functions of chaperone proteins (chaperonins) in the RER.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Translation and Protein Processing 1&2 Elitsa Ananieva, PhD Learning Objectives 1. Describe the process of protein translation and recognize the molecular players and their roles in this process. a. What is the purpose of amino acid activation and how does it work? b. What are the rules behind codon and anti-codon-pairing? c. What are the main features of the genetic code? d. What is the role of inosine (tRNA) in codon and anti-codon pairing? e. Describe the steps (including molecular players) that lead to initiation, elongation, and termination of translation. f. What are polysomes and how do polysomes work? 2. Explain the impact of antibiotics and toxins on bacterial and human protein translation. a. What is the mode of action or targets of: streptomycin, tetracycline, chloramphenicol, erythromycin, neomycin/gentamycin? b. What are the biochemical and clinical features of gray baby syndrome? c. What is the mode of action or targets of diphtheria toxin and ricin? 3. Explain the importance of posttranslational processing for proper protein function and export to organelles and membranes. Identify mechanisms to dispose of damaged or regulatory proteins. Recognize how dysfunctional protein processing can contribute to a disease state. a. Compare N- and O-linked glycosylation in terms of: (1) location, (2) sugar monomers used, (3) amino acids (amino acid groups) involved in the binding of sugar monomers, and (4) protein folding. Give examples of N- and O- glycosylated proteins. Explain the role of dolichol phosphate for the N-linked glycosylation of proteins? b. What is the function of chaperones? c. List different types of lipid modifications and other groups additions. d. What are the biochemical and clinical features of Menkes disease? e. Briefly explain protein trafficking to the mitochondria. f. Compare lysosomal and proteasomal degradation. What is the role of poly- ubiquitination and E1, E2, E3 enzymes during protein degradation? g. What are the biochemical and clinical features of: (1) cystic fibrosis; (2) I-cell disease; (3) α1-antitrypsin deficiency.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Overview of Translation The next step in gene expression is the translation of the nucleotide sequence of mRNA into the amino acid sequence of a protein where: mRNA is the working copy of the gene. three nucleotides (triplet or codon) of DNA that are transcribed into mRNA serve as a genetic code that specifies an amino acid in the protein chain. tRNA serves as an adaptor molecule that couples the codons in mRNA with the amino acids, they specify, thus aligning them into the polypeptide chain of a protein. rRNA together with proteins form the ribosomes where translation takes place. Translation is thus a highly coordinated process that requires mRNA, tRNA, rRNA, enzymes, and other protein factors to succeed. Features of mRNA, tRNA, and Ribosomes Features of mature mRNA Mature mRNA, that is transported into the cytoplasm and is ready to enter translation, has the following structural features (Fig.1): Fig.1. Structure of the mature mRNA. In this structure: AUG is the start codon situated at the 5’ end of mRNA. There are examples of mRNAs carrying more than one start codons; in this case folding of mRNA prevents the reading from the “false start codons”. UAG is the stop codon at the 3’ end before the poly A tail signal. The region of mRNA, that encodes for a protein, is also known as open reading frame (ORF). Features of tRNA tRNAs are transcribed by Pol III. Once they are synthesized, they travel to the cytoplasm where they combine with the appropriate amino acid and take part in translation. Structure of tRNA The structure of tRNA resembles a cloverleaf and has around 80 nucleotides. The most important structural features of tRNA are shown in Fig.2:  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Fig.2. The structure of tRNA. In this structure: The acceptor arm (CCA), situated at the 3’ end of tRNA, is the site where activated amino acid is attached. The anti-codon arm is where the tRNA anneals with the 3-base codon of mRNA. The anti-codon is complementary and antiparallel to the codon of the corresponding mRNA. Additionally, tRNA has a D loop that contains dihydrouridine (modified nucleotide) and TψC loop that contains ribothymidine and pseudouridine. Amino acid activation The attachment of an amino acid to its corresponding tRNA molecule is an energy consuming process, known as amino acid activation, and is shown in Fig.3: Fig.3. Amino acid activation. Figure modified from USMLE Step I Kaplan, 2011. Enzyme, known as aminoacyl tRNA synthetase, catalyzes the attachment of amino acid to an tRNA molecule: o the enzyme requires two high energy bonds from ATP. o the enzyme attaches the amino acid to the 3’ end of the tRNA molecule.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 o the bond between the amino acid and tRNA contains high energy that is used later during the formation of a peptide bond thus helping link amino acids in a protein chain. o there is a specific aminoacyl tRNA synthetase for each of the 20 amino acids to be incorporated into protein; therefore, there are 20 different aminoacyl tRNA synthetases. Regulation. The aminoacyl tRNA synthetase possesses self-checking function against incorrect pairing of an amino acid and tRNA. Symbols. the aminoacyl-tRNA is written as aminoacyl-tRNAname of amino acid (for example: aminoacyl-tRNAAla or Ala—tRNAAla). Codon anti-Codon pairing The codon anti-codon pairing includes the following rules (Fig.4): The anti-codon sequence of tRNA is complementary and antiparallel (runs in the opposite direction) to the codon, carried by mRNA. The genetic code (as shown in the Table) is represented by 64 possible codon sequences (since 3 bases make one codon). 61 codons encode for amino acids while 3 encode for stop codons (UAA, UGA, UAG). The start codon is always AUG and it encodes for methionine (Met) in eukaryotes. Each codon specifies only one amino acid; thus, the code is unambiguous. For example, the codon Fig.4. Illustration of anti-codon codon paring (top). Table CCC encodes proline of the genetic code (bottom). Do not memorize the (Pro) only! codons.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 There is more than one codon that codes for a given amino acid; thus, the code is degenerate. For example, Pro is coded by CCC, but also by CCU, CCA, and CCG. Note, that the first two bases are the same but the third one is different. The code is universal. The code is “comaless” and “non-overlapping” meaning that there are no spaces between the codons and two codons do not overlap with each other. There are around 50 different tRNA molecules, does this mean that there are not enough tRNA molecules for each of the 64 codon combinations? No, because some tRNAs can pair with more than one codon (“wobble hypothesis”). According to the wobble hypothesis, position one in the anti-codon is flexible and can pair with different nucleotides. tRNA contains inosine that can pair with A, C, or U. Features of Ribosomes Ribosomes are the protein synthesizers. They are composed of rRNA and proteins forming large complexes that are measured in sedimentation constants “S”. The different types of ribosomes are described in the Table (for illustration purpose only): Main characteristics of ribosomes are: In the nucleolus, Pol I transcribes the eukaryotic rRNA as a single 45S rRNA, which is cleaved later into 28S and 18S, and 5.8S rRNAs. The 5S rRNA is transcribed by Pol III from a separate gene. The rRNAs then combine with proteins in the nucleolus to form the small and large ribosomal subunits. During protein synthesis in the cytosol, the two subunits join to form the whole 80S ribosome. Note that “S” is a function of both size and shape and therefore the numbers are not additive- 40S plus 60S subunits give rise to 80S ribosome (Fig. 5). Humans have mitochondrial ribosomes that are different from the cytosolic ribosomes described above; They are important for protein synthesis inside the mitochondria. Prokaryotic ribosomes are different from the cytosolic and mitochondrial ribosomes; This is important in terms of the use of antibiotics that can selectively target bacterial, but not human ribosomes during treatments.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Fig.5. The small and large subunits of the ribosome. Steps in Translation Translation takes place in the cytoplasm and is divided into three stages: initiation, elongation, and termination, as described below. Initiation The initiation comprises 2 important steps: (1) The assembly between the small subunit, tRNA carrying methionine, and mRNA. This assembling is guided by initiation factors, which, in eukaryotes, are known as eIFs (eukaryotic initiation factors). (2) The large subunit binds the assembled complex between the small subunit, tRNA, and mRNA, thus concluding the initiation phase. In greater detail, the initiation proceeds in the following order (Fig.6): Eukaryotic initiation factor 2 (eIF-2) binds GTP and becomes activated. Activated eIF-2 then binds met-tRNAmet (initiator tRNA); the initiator tRNA carrying Met is the first tRNA to be incorporated. It recognizes the AUG codon of mRNA. The complex between eIF-2 and met-tRNAmet is known as the ternary complex. The ternary complex then binds the small ribosome subunit; the latter is bound by two additional eukaryotic initiation factors, eIF-1 and eIF-3. Soon after, another eukaryotic initiation factor (eIF-4) directs this complex toward the 5’ end of mRNA by binding the 5’ cap. This complex is known as the pre-initiation complex. Once the large ribosomal subunit binds the pre-initiation complex and the eIFs come off, the initiation complex is formed. The fully assembled ribosome has three important sites (shown in Fig.7) o P (peptidyl site), occupied by the methionyl-tRNAmet in the end of the initiation process. o A (acceptor site) where the incoming aminoacyl-tRNAs bind one by one during elongation; this site is unoccupied during initiation. o E (ejection site) necessary for the removal of tRNAs after a peptide bond is formed; this site is unoccupied during initiation.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Fig.6. Initiation of translation and the role of initiation factors (eIFs) during the formation of the initiation complex. Elongation There are three important steps during elongation: (1) Binding of aminoacyl-tRNAs (while delivering amino acids) at the A site in a subsequent manner. (2) Formation of a peptide bond between two adjacent amino acids. (3) Translocation of the peptidyl-tRNA to the P site. In greater detail, elongation proceeds in the following order: The initiator tRNA, carrying Met, occupies the P site of the ribosome. The eukaryotic elongation factor eEF-1 helps to deliver a second aminoacyl- tRNA to the A site. This step requires energy from GTP hydrolysis. The peptidyltransferase activity of the ribosome (large subunit) catalyzes the formation of a peptidyl bond between the first and the second amino acids (Met and Ala in the example in Fig. 7). Translocation: The ribosome moves one codon down the mRNA in 5’-3’ direction, thus synthesizing a protein from the amino to the carboxyl terminus; the tRNA with  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 two amino acids connected in a peptide chain is now called peptidyl-tRNA. It moves to the P site, while the uncharged tRNA, originally in the P site, moves to the E site for ejection. This process is assisted by the eukaryotic elongation factor eEF-2 and requires energy from GTP. The ribosome is now ready for the next amino acid to be delivered by the corresponding aminoacyl-tRNA. Fig.7. The elongation of the peptide chain. Termination Termination (Fig.8) occurs as follows: it starts when a stop codon (UGA, UAG, or UAA) enters the A site. a eukaryotic release factor (eRF), bound to GTP, pairs with the stop codon of mRNA. GTP is used to supply energy for the release of the newly synthesized peptide chain from the ribosome. Ribosomal subunits dissociate from each other and mRNA; tRNA and protein are released as well. Fig.8. Overview of termination of translation.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Polysomes Several ribosomes can attach to a single mRNA at any given time forming the polysomes (Fig.9). Each polysome carries a polypeptide chain, which grows bigger as the ribosome approaches the 3’ end on mRNA. Thus, the polysomes can simultaneously read a single mRNA molecule to synthesize the same protein in multiple copies. The polysomes can be either free in the cytoplasm or attached to the rough endoplasmic reticulum (RER). Fig.9. Polysomes. Inhibition of Protein Synthesis Clinical Correlation: Antibiotics The following antibiotics can inhibit protein synthesis on prokaryotic (bacterial, 70S) ribosomes and therefore are used to treat a variety of bacterial infections: Streptomycin It binds the 30S bacterial ribosomal subunit and causes misreading of mRNA thus preventing the formation of the initiation complex. Tetracycline It binds the 30S bacterial ribosomal subunit but inhibits the binding of aminoacyl- tRNA to the A site. Chloramphenicol It inhibits the peptidyl transferase activity of the 50S bacterial ribosomal subunit. Erythromycin It binds the 50S bacterial ribosomal subunit and prevents the translocation during the elongation phase. Neomycin and gentamycin They bind the 30S bacterial ribosomal subunit and cause mistranslation of the mRNA codons (incorrect aa-tRNAs may be incorporated). Clinical Correlation: Gray baby syndrome Definition:  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Gray baby syndrome is an adverse reaction to chloramphenicol. Chloramphenicol is a man-made antibiotic initially used for the treatment of typhoid fever. The first case of a fatal adverse reaction to chloramphenicol was reported 12 years after chloramphenicol discovery (first discovered in 1947). Biochemical features: Since mitochondrial ribosomes are somewhat similar to the prokaryotic ribosomes, chloramphenicol can inhibit the eukaryotic mitochondrial protein synthesis resulting in impaired electron transport and cellular toxicity. Chloramphenicol can also displace unconjugated bilirubin from albumin, causing brain damage (kernicterus, see heme lecture) and eventually death if left untreated. Clinical features: o vomiting and hypothermia. o ashen-gray skin discoloration (blue lips, blue nail beds). o premature infants and neonates are at the highest risk from chloramphenicol exposure due to their decreased hepatic and renal function (inability to detoxify the antibiotic). Clinical Correlation: Toxins Toxins are chemicals that can interfere with the eukaryotic protein synthesis thus causing human diseases: Diphtheria toxin o Synthesized by Corynebacterium diphtheriae (more specifically its phage genes). o Fragment from the toxin (A fragment) inhibits eEF-2 thus preventing translocation during elongation. The inhibition involves ADP- ribosylation of eEF-2 (discussed later). o The toxin causes diphtheria (severe sore throat and fever) that can be lethal. Ricin o It is a glycoprotein with N-glycosidase activity. o This enzyme catalyzes the cleavage of an adenine base from the 28S rRNA of the large eukaryotic ribosomal subunit. o The cleavage inhibits protein synthesis due to a loss of binding of initiation and elongation factors to the large ribosomal subunit. o Ricin can be extracted from the oil of castor beans. Shiga toxin (verotoxin) o Synthesized by Shiga dysenteriae or E.coli (more specifically its phage genes). o The drug inactivates the 28S rRNA and the 60S ribosomal subunit via similar mechanism as ricin.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Protein Processing Upon completion of protein translation, the polypeptide chain (or chains) of a protein undergoes one or more of the following steps: Folding Post-translational processing (or modifications) Transportation to the correct cellular compartment Protein Folding Folding occurs as soon as the polypeptide chains emerge from the ribosomes and includes the following: Formation of three-dimensional protein conformations that are essential for biological activity; they were covered by Dr. Wilson. Folding of soluble (cytoplasmic) proteins usually occurs immediately and they spontaneously obtain their correct conformation. Folding of other proteins, however, requires the assistance of proteins, known as chaperones: o Chaperones bind the hydrophobic regions of partially folded peptides and guide their correct folding. o They are found in the cytoplasm and the endoplasmic reticulum, as both locations are places of protein folding. o Heat shock proteins (HSPs) are a group of chaperone proteins: ▪ they function to repair damaged proteins due to heat or stress. ▪ HSPs are highly expressed in some cancers leading to drug resistance. ▪ mutations in HSPs can cause misfolding disorders (Charcot Marie Tooth disease). Post translational modifications After translation, proteins may be subjected to posttranslational modifications, such as chemical group addition (carbohydrate, lipid, or other), cleavage of a portion of the protein etc. Proteins that commonly undergo posttranslational modifications are listed below: Proteins destined to organelles are targeted by glycosylation and other modifications necessary for proper transportation. Examples: lysosomal proteins (glycosylation), nuclear proteins (addition of basic amino acids), mitochondrial proteins (signal sequence). Membrane proteins need to be anchored to the membranes. Lipid addition is required to attach these proteins to membranes (examples: Ras, GRB2). Inactive proteins. Some proteins are synthesized in an inactive form and further modifications ensure that they become active. Examples: protein phosphorylation (signaling proteins), proteolysis (digestive enzymes), hydroxylation of proline (cross-linking of collagen).  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Misfolded proteins. Proteins that are misfolded or incorrectly modified are recognized and moved into the cytoplasm for destruction. Carbohydrate addition: Glycosylation Description and Importance: This is a covalent addition of carbohydrate chains to proteins. Glycosylation is important for the formation of recognition sites that direct protein trafficking (lysosomal proteins) or are used during protein-protein interactions. Glycosylation increases the solubility, stability, and size of proteins that are glycosylated. Proteins that are commonly glycosylated: antibodies, protein hormones, growth factors, cytokines, lysosomal proteins. Glycosylation is catalyzed by glycosyltransferases. o They transfer a sugar (monosaccharide) from a sugar nucleotide (sugar donor) to a protein (acceptor). commonly used sugar monomers are mannose (Man), glucose (Glc), N-acetylglucosamine (GlcNAc), fucose (Fuc), galactose (Gal), N- acetylgalactosamine (GalNAc), sialic acid. commonly used sugar donors are UDP-N-acetylglucosamine, GDP- mannose, UDP-galactose, CMP-sialic acid. o They catalyze a reaction that includes the formation of glycosidic bonds, and each glycosyltransferase is specific for both the sugar donor nucleotide and the acceptor molecule. Types of glycosylation There are two types of glycosylation (1) N-linked glycosylation and (2) O-linked glycosylation. N-linked glycosylation This is the addition of sugar monomers (in the form of oligosaccharide) to the amino acid asparagine of proteins undergoing glycosylation (Fig.10). Fig.10. N-linked glycosylation of a protein. N-acetylglucosamine is attached to the amide group of asparagine. N-linked glycosylation occurs in two steps: Step (1) Building a Universal Oligosaccharide: A lipid known as dolichol, embedded in the ER membrane, serves as a platform to build the universal oligosaccharide. Note that, dolichol must be phosphorylated to assist with N-linked glycosylation (dolichol phosphate).  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 The first sugar monomer to be attached to dolichol is always N- acetylglucosamine (GlcNAc)!!! This process occurs in the endoplasmic reticulum (ER) and is catalyzed by glycosyltransferases attached to the cytosolic and luminal faces of ER. The enzymes add sugar monomers until the universal oligosaccharide is formed. Next, the oligosaccharide is transferred from the dolichol phosphate to the nascent polypeptide chain (see Fig. 11) Fig.11. N-linked glycosylation. Formation of the universal oligosaccharide in ER. Step (2) Specific modifications of the universal oligosaccharide it takes place in the Golgi apparatus (Fig. 12). It leads to either high mannose type (containing mannoses) or complex type (containing sialic acid, fucose, N-acetyl-glucosamine, galactose etc.,). the diversity of glycoproteins is thus determined by different combinations of sugar monomers and different types of glycosidic bonds between them. o an example of N-glycosylated protein is the chloride channel membrane protein (CFTR). Disorder in N-glycosylation of CFTR causes misfolding of CFTR and leads to the development of cystic fibrosis (see it in clinical correlation of Protein Processing and Degradation). Fig.12. N-linked glycosylation. Unique modifications leading to either high mannose type or complex type glycosylated proteins. Figure modified from Dr. Schmidt’s original figure.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 O-linked glycosylation It starts with the addition of a sugar monomer to the hydroxyl group of either serine, threonine, or tyrosine residues of proteins to be glycosylated (Fig.13). Fig.13. O-linked glycosylation. N- acetylglucosamine is attached to the hydroxyl group of either serine, threonine, or tyrosine. Next, other sugars are added, such as glucose, galactose, fructose, sialic acid. Donors of sugar monomers are activated nucleotides, same as the ones described above. This type of glycosylation occurs on fully folded proteins only. It takes place in the Golgi apparatus not ER. Clinical Correlation: ABO Blood Type The human A, B, and O blood-group antigens can exist as both glycoproteins and glycolipids on the surface of erythrocytes (Fig.14): Each antigenic determinant consists of a short oligosaccharide chain (Glc-Gal- GlcNAc-Gal-Fuc) attached to either a lipid or serine/threonine residues of a protein (O-linked glycosylation). Fig.14. O-linked glycosylation of the ABO blood types. The A antigen contains one additional sugar monomer (N-acetylgalactosamine, GalNAc), attached to the outer galactose residue. People with type A blood have a specific GalNAc transferase enzyme that adds the extra N-acetylgalactosamine.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 The B antigen has one additional galactose (Gal) residue, attached to the outer galactose; People with type B blood have a specific Gal transferase enzyme that adds the extra galactose. The O antigen has NO additional monomers attached to the outer galactose. People with O blood type do not express either enzyme, while people with AB blood type express both transferases and synthesize both the A and B antigens. The table summarizes the relevance of the A, B, and O antigens to blood transfusions. For example, people lacking Gal transferase cannot synthesize the B antigen (blood types A and O). If blood type B or AB is transfused into a person with blood type A or O, this person will produce anti-B antibodies that will bind to the transfused erythrocytes and trigger an immune destruction. Thus, blood-group typing, and appropriate matching of blood donors and recipients is required in all transfusions. Lipid addition Lipid groups are added to proteins that associate with membranes allowing tethering of the proteins: Palmitoylation is the addition of palmitic acid to cysteine residues of proteins (Fig.15): Fig.15. Palmitoylation of membrane proteins. Prenylation is the addition of isoprenoids to cysteine residues of proteins (Fig.16): Fig.16 Prenylation of membrane proteins.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Myristylation is the addition of myristic acid to N-terminal glycine residues of proteins (Fig.17): Fig.17. Myristylation of membrane proteins. Addition of other chemical groups Phosphorylation is the addition of a phosphate group to serine, threonine, or tyrosine residues of a protein, which serves to either activate or inactivate a protein (Fig.18): Fig.18. Phosphorylation of proteins. Acetylation is the addition of an acetyl group to lysine residues of proteins. Histones are modified by acetylation of their lysine residues, which is important for the regulation of gene expression (Fig. 19). Fig.19. Protein acetylation. ADP-ribosylation is the addition of ADP-ribose to arginine or glutamine residues of proteins (Fig. 20). Diphtheria uses ADP-ribosylation to inhibit the function of eEF-2. Fig. 20. ADP-ribosylation of proteins.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Proteolysis. This is not the addition, but the cleavage of peptide bonds to remodel and activate proteins (for example, pepsinogen to pepsin or proinsulin to insulin). Hydroxylation of prolyl and lysyl residues in the molecule of collagen gives rise to hydroxyproline and hydroxylysine. Hydroxyproline (Fig.21) is a unique amino acid in the molecule of collagen. It is important for the proper formation of collagen fibrils. Failure to hydroxylate proline and lysine leads to disorders in collagen processing. Fig.21. Hydroxylation of proline. Clinical Correlation: Disorders in Collagen Biosynthesis and Processing Scurvy: results from insufficient vitamin C uptake. Vitamin C is a co-factor of prolyl and lysine hydroxylases, the enzymes that hydroxylate proline and lysine. covered by Dr. Wilson Ehlers Danlos syndrome: collection of defects in the synthesis and processing of collagen. covered by Dr. Wilson Menkes disease (OMIM# 309400) Biochemical features Mutations in the ATPase copper transporting alpha (ATP7A) gene cause Menkes disease. This gene encodes a protein that regulates copper levels in the body. When ATP7A is mutated, copper accumulates in the small intestine and kidneys, while the brain and other tissues have copper deficiency. This affects numerous copper-containing enzymes including lysine hydroxylase that is responsible for lysine hydroxylation in collagen. The occurrence of Menkes is rare, with an estimated incidence of 1 in every 250,000 live births. Clinical features o severe growth failure o profound neurodegeneration o osteoporosis o depigmented hair o hypotonia  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Protein targeting to the correct cellular compartment Protein targeting for delivery to peroxisome, lysosomes, or nucleus Proteins destined to intracellular organelles, such as peroxisomes, lysosomes, and the nucleus, are targeted by the addition of sugar (lysosomes) or amino acids (nucleus, peroxisomes), the latter are short signal sequences (motifs) at the N-terminus of the peptide chain but are subsequently cleaved after the protein reaches final destination (Fig. 22). Fig.22. Targeting of proteins destined to organelles such as the peroxisomes, lysosomes, ER, and the nucleus. Protein targeting for delivery to mitochondria Proteins destined to the mitochondria are pre-synthesized as larger proteins. The process of their transportation to the mitochondria comprises the following steps: These proteins are synthesized in the cytosol as large preproteins (sometimes called precursors) with N-terminal leader sequence (pre-sequence). They are assisted by chaperones (Hsp70) that help deliver them to the mitochondria. The leader sequence binds to a receptor on the outer mitochondrial membrane. A protein complex between TOM and TIM (translocases of the outer and inner mitochondrial membranes) provides a channel for the transportation inside the mitochondrion. Matrix proteases (known also as matrix processing proteinases, MPP) then cleave off the leader sequence and a mature protein is released inside the mitochondrion (more chaperones help during this process). Clinical correlation of TIM deficiency Deafness dystonia syndrome (OMM#300356) is caused by mutations in TIM leading to dysfunctional mitochondria. This disorder impacts energy dependent tissues, such as the nerves and muscles Protein Degradation Protein stability varies widely; hence the half-life of proteins can range from minutes to years. Certain groups of proteins are more often targeted for protein degradation then others: Regulatory proteins. These proteins experience rapid protein turnover to allow their levels to change quickly in response to external stimuli. Damaged proteins. They are recognized and rapidly degraded to avoid the accumulation of dysfunctional proteins, which ultimately may lead to diseases.  ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Mechanisms of protein degradation There are two different mechanisms of protein degradation in eukaryotes: lysosomal degradation (1) and degradation via the proteasome (2). (1) Lysosomes: They function in association with the process of autophagy (cell digestion). Lysosomal degradation is nonspecific and may include extracellular and intracellular proteins. The lysosomes contain hydrolytic enzymes (lipases, proteases, glycosylases) that can degrade different cellular molecules not just proteins. The lysosomal enzymes are post-translationally modified by N- glycosylation with mannose-6-phosphate. This modification is critical for their direction to the lysosomes. (2) Proteasomes and ubiquitin This is the degradation pathway for cytoplasmic proteins. Many cytoplasmic proteins are subjected to proteasomal degradation as a part of regulatory mechanisms (refer to our Cell Cycle lecture). Cytosolic proteins that are not properly folded are commonly targeted for proteasomal degradation. Proteins targeted for proteasomal degradation are marked with ubiquitins. Ubiquitins undergo activation, conjugation, and ligation prior to becoming protein targets. An enzyme, E1 ubiquitin-activating enzyme, activates ubiquitins, a second enzyme, E2 ubiquitin-conjugating enzyme, conjugates ubiquitins to polyubiquitin chains, while a third enzyme, E3 ligase, ligates the polyubiquitin with the targeted protein. There are multiple isoforms of E3 with specificity toward a certain class of proteins. Polyubiquitinated proteins are then destroyed via the proteasomes. Proteasomes are large cytosolic protein complexes with multiple protease activities, needed to digest the protein targets. Only polyubiquitinated proteins are subject of degradation. A single ubiquitin attachment to a protein is not a signal for degradation. Clinical correlation of Protein Processing and Degradation: Cystic Fibrosis (OMIM#219700) Biochemical features: Cystic fibrosis is a rare genetic disorder that commonly results from mutations in a gene encoding a chloride channel membrane protein, which is most commonly refer to as the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation causes a deletion of phenylalanine at position 508 that interferes with the folding and processing of CFTR. As a consequence, CFTR is subjected to proteasomal degradation. Clinical features: A loss of CFTR affects the cells that produce mucus and digestive juices causing stickiness of these fluids and thus plugging of ducts and passageways. ANANIEVA TRANSLATION AND PROTEIN PROCESSING 2024 Symptoms include cough, repeated lung infections, inability to gain weight, and fatty stools. Clinical correlation of Protein Processing and Degradation: I-cell Disease (OMIM #252500) Biochemical features: I-cell disease (also known as mucolipidosis II) is caused by impairment in the transfer of a phosphate to mannose during the posttranslational modification of lysosomal enzymes. As a result, the lysosomal enzymes cannot reach their final destination. In the absence of lysosomal enzymes, the lysosomes are nonfunctional and stacked with undegraded proteins or other waste. On genetic level, the impairment in the posttranslational processing of the lysosomal enzymes is due to mutations in the GNPTA gene, which encodes the enzyme UDP-N- acetylglucoseamine-1-phosphotransferase that is involved in the synthesis of mannose-6-phosphate. Clinical features: o Fibroblasts with inclusion (I) bodies (thus I–cell disease) containing undigested waste products. The I-bodies appear as abnormal vacuolization or inclusions especially in the skeletal system. o Secretion of lysosomal enzymes in the serum o Bone fractures and slow motor abilities o Growth retardation and possible death Clinical correlation of Protein Processing and Degradation: α1-Antitrypsin Deficiency (OMIM #613490) Biochemical features: This disorder results from mutations in some of the allelic variants (Z, S) of the gene encoding α1-antitrypsin protein. This protein is normally expressed in the liver and then secreted in the blood stream where in inhibits proteases during inflammatory response. Patients who express the M allele of the gene produce normal α1-antitrypsin and do not develop disease. Mutations in the gene [allelic variants (Z, S)] result in misfolding and accumulation of aggregates in ER. Clinical features: o Liver cirrhosis o Chromic pulmonary disease  FS1 28 / MSK 09 – SER, Mitochondria, & Energy Storage – Fall 2024 Dr. Sarah Werning – [email protected] Lab 06 is associated with this lecture (in part) Recommended Readings GTH – note: many SER functions are organ-specific, so these jump all over the book Ch 2 (Cytoplasm): Endoplasmic Reticulum; Mitochondria; Glycogen; Lipids; Ch 8 (Muscle): T Tubules & Sarcoplasmic Reticulum; Ch 13 (Endocrine System): Suprarenal Cortex (stop at Zona Glomerulosa); Ch 18 (Digestive System: Glands): Hepatocyte Organelles and Inclusions; Histophysiology of the Liver (stop at Immune Function) o Relevant figures (lecture): Figs 2.5, 2.6, 2.13, 2.29, 2.30, 18.27, 18.30, 18.31 GCA – Ch 1 (The Cell): Mitochondria; Endoplasmic Reticulum; Plates 1-5 (Typical Cell, Electron Microscopy), 1-6 (Nucleus & Cytoplasm, Electron Microscopy), 1-7 (Nucleus & Cytoplasm, Electron Microscopy), 1-9 (Mitochondria, Electron Microscopy) o All plates listed above are helpful for Lab 6 W – Ch 1 (Cell Structure and Function): Energy Production and Storage; Lipid Biosynthesis; Figs 1.7a, 1.8, 1.18, 1.19, 1.20, 1.23, 1.24; Ch 4 (Supporting/Connective Tissues): Fig 4.18 only; Ch 17 (Endocrine System): Fig 17.16c only o All figures listed above are helpful for Lab 6 BRS – Ch 1 (Cell): sections VII.A.3, 4, 5 (SER, Peripheral ER, Mitochondria) only Lecture Objectives See last 2 pages for example open-ended study questions for each lecture objective. After reviewing this lecture, the learner should be able to: 1. Relate the structure of the smooth endoplasmic reticulum (SER) to its functions. 2. Relate the structure of the mitochondrion to its functions. 3. Describe how energy is stored in glycogen granules and lipid droplets. 4. Describe examples of clinical significance relating to the SER, mitochondria, glycogen granules, & lipid droplets. Lecture Outline I) Smooth Endoplasmic Reticulum A) Main Functions of the SER - many SER functions are organ/tissue-specific! synthesize lipids  ‒ mainly phospholipids & cholesterol, but can make most major classes of lipids ‒ assembles & repairs most of the membrane lipids used in the cell ‒ main membrane lipids = phospholipids, cholesterol ‒ others would be glycolipids, sphingolipids (e.g.) ‒ synthesizes & releases steroid hormones ‒ using cholesterol as a source ‒ note: SER is not the only organelle that makes lipids! most fatty acids & triglycerides are synthesized in the cytosol mitochondria & peroxisomes can make certain membrane lipids RER & Golgi help modify lipids; RER may have limited lipid synthesis ability drug detoxification (liver cells) – only some drugs! e.g. barbiturates & alcohol assists in glucose metabolism (mainly in liver cells) calcium (Ca2+) storage, release, & recapture (especially skeletal & cardiac muscle) B) SER Structure in most cells, SER < RER. SER is only extensive in cells that are specialized for a major SER function (e.g. liver cells, muscle cells, steroid-synthesizing cells) SER is an interconnected network of cisterns (cisternae) membrane-bound (single membrane) lumen & membrane of SER cisterns are continuous with those of RER SER cisterns are more tubular & have distinct branches differs from RER, which is stacks of flattened sacs SER cisterns lack ribosomes  “smooth” relative to RER SER is often associated with small lipid droplets (steroid-synthesizing cells have many) imaging: TEM: cisterns are thin tubes, may be closely packed & twist around each other ⇒ often a transitional area “grades into” RER LM: SER not resolvable in LM, but cytoplasm is more acidophilic in cells with lots of SER (less RER/ribosomes); sometimes lipid droplets are visible in LM in steroid- secreting cells (paler & “foamy” appearance) C) SER Synthesis of Membrane Lipids phospholipids ‒ synthesis occurs on the cytosolic monolayer of the SER membrane ‒ necessary enzymes are associated with the cytosolic monolayer ‒ but the metabolites are all in the cytosol ‒ precursors are assembled at the interface of SER & cytosol ‒ precursors brought into cytosolic monolayer & assembly is finished there ‒ this introduces asymmetry in phospholipid content between monolayers ‒ now there is a surplus of phospholipids on cytosolic monolayer 1. need a bilayer  need to add some to the luminal monolayer  ‒ flippases & floppases (phospholipid translocator proteins) transfer phospholipids from cytosolic monolayer to luminal monolayer 1. scramblase (most active flippase in SER) can only transfer choline-containing phospholipids to the luminal monolayer 2. other kinds of phospholipids are over-represented on cytosolic side ‒ phospholipid asymmetry is maintained after transfer ‒ SER sends newly-formed bilayers to RER via lateral transfer 1. their membranes are continuous ‒ SER sends newly-formed bilayers to Golgi, lysosomes, & cell membrane via transfer or secretory vesicles 1. the new bilayer buds off SER to form a vesicle & merges with destination membrane 2. these organelles have very few flippases, so the asymmetry persists ‒ unclear how mitochondria & peroxisomes maintain phospholipid asymmetry ‒ do not receive phospholipids from SER in vesicles ‒ mitochondria synthesize cardiolipin (mt-exclusive phospholipid) ‒ mitochondria & peroxisomes get most phospholipids via water-soluble phospholipid transfer proteins 1. choose 1 specific phospholipid molecule from cytosolic monolayer in SER & carry it to mitochondrion or peroxisome 2. can only insert phospholipids in cytosolic monolayers though 3. there must a flippase or floppase present, but identity is unknown glycolipids ‒ enzymes essential for glycosylation of glycolipids are present on the lumen side of SER, RER, & Golgi  all 3 organelles have some capability ‒ this is why all glycolipids & glycoproteins have sugar residues on the luminal surface of organelle membranes ‒ and why sugar residues on the extracellular side of cell membranes ‒ often: SER assembles lipid component  travels to RER via lateral transfer ‒ RER sends to Golgi via vesicle  modifies by adding sugars ‒ Golgi sends to final destination via secretory/transport vesicles SER also synthesizes ceramide (type of sphingolipid used to make sphingomyelin) SER also synthesizes cholesterol for membranes D) Other SER functions SER is main calcium (Ca2+) storage site in cell (regulates, releases & recaptures) ‒ Ca2+ critical for cell signaling, neurotransmitter release & muscle cell contraction ‒ SER very prominent in striated muscle cells  sarcoplasmic reticulum ‒ cell damage occurs if cytosolic Ca2+ levels remain elevated steroid hormone synthesis SER very prominent in steroid-secreting cells (adrenal glands, gonads)  glucose metabolism glycogenolysis = enzymatic breakdown of glycogen via sequential removal of glucose molecules ‒ enzyme on lumen side of SER membrane completes the last step ‒ Clinical relevance: glycogen storage diseases ⇒ if this enzyme or its transporter lose function, the cell can’t break down glycogen  glycogen accumulates in liver & kidney cells gluconeogenesis = other main supply of glucose to body during fasting ‒ enzyme on lumen side of SER membrane, primarily in liver & renal cortex drug detoxification ‒ enzymes that metabolize alcohol and certain drugs (e.g., phenobarbital) are located on the lumen side of SER membrane, primarily in liver ‒ Clinical relevance: prolonged alcohol/drug use leads to increased tolerance tolerance: repeated use leads to shorter therapeutic response, so larger doses must be used to achieve the desired effect mechanism: compensatory increase in SER volume, and increase in the enzymes that metabolize drugs/alcohol ‒ more enzymes  faster metabolism (= faster detoxification)  wears off faster or effect is muted  must take more to get same effect  more or less a positive feedback loop that keeps increasing sER effects of increased SER volume can be really dramatic! source: The Gazette ‒ July 2012: 24-yo man arrested in North Liberty, IA on suspicion of DUI. Breathalyzer on the scene: BAC = 0.486%; blood test 1 hr later: 0.627% 1. legal limit in Iowa (& most states) for = 0.08% 2. most people black out ~ 0.20% 3. lethal dose is variable, between 0.30 and 0.40% 4. 0.627% is the equivalent of ~35 drinks in 4 hours II) Overview/Review of Energy Production & Storage all cells depend on a continuous energy supply for metabolic functions ‒ derived from sequential breakdown of organic molecules during the process of cellular respiration ‒ energy ultimately stored in a readily available form: ATP molecules main substrates = simple sugars (esp. glucose) & lipids (esp. fatty acids) cellular respiration of glucose ‒ glycolysis begins in cytosol: glucose  pyruvic acid & ATP (small amount) ‒ pyruvic acid diffuses into mitochondria in presence of O2, degraded into CO2 + H2O + lots of ATP cellular respiration of fatty acids (FAs) ‒ FAs pass directly to mitochondria, which degrade them via β-oxidation in presence of O2, degraded into CO2 + H2O + lots of ATP  mt can only break down short, medium, & long chain FAs; very long chain FAs are into shorter chains by peroxisomes can happen in the absence of oxygen (anaerobic respiration) but this is less efficient if there is excess fuel, most cells convert it for storage ‒ glucose  glycogen ‒ fatty acids  triglycerides ‒ amounts of each differ by cell type: neurons: very little of either most of body’s limited storage of glycogen is in liver & muscle cells almost unlimited storage available for triglycerides in fat cells III) Mitochondria (single = mitochondrion) I abbreviate mitochondrion & mitochondria as mt throughout this section mobile “power generators” that migrate around the cell to supply energy where needed A) Main Functions break down short, medium, & long chain fatty acids via β-oxidation produce most of the cell’s ATP (mt = principle organelle of cellular respiration) ‒ via oxidative phosphorylation ‒ aerobic  require a constant supply of oxygen ‒ large #s in metabolically active cells decide whether the cell lives or dies ‒ sense cellular stress ‒ trigger release of enzymes that initiate apoptosis temporary storage, release, & recapture of calcium (Ca2+) B) Location & number throughout cytoplasm, but tend to concentrate in areas of high metabolic activity mobile  travel via motor proteins on microtubules vary in # depending on metabolic activity of cell ‒ only cells that lack mt = red blood cells & terminal keratinocytes ‒ inactive cells have a few ‒ liver cells have 1000+ (22% cell volume) ‒ cardiac muscle cells: up to 40% cell volume number of mt in a cell is always changing ‒ mt division is not synchronized with cell division ‒ mt # increases by fission, which happens throughout the cell cycle fission = splitting; mt fission is very similar to bacterial cell fission ‒ mt # decreases by fusion of mt or autophagy of mt ‒ increases in response to exercise, decreases in response to sedentary life C) Structure shape is variable: ovoid, spherical, threadlike, coiled ‒ also they change shape & in some cells they fuse  individual mt are too small to see in LM: ~1 μm wide & 5-10 μm long 2 membranes (2 +/- concentric bilayers) separated by an intermembranous space autonomous double-stranded circular DNA (mtDNA) ‒ < 1% total DNA, but many copies  < 1x10-5 #nucleotides vs nucDNA ‒ all descend from maternal mt present in the egg at fertilization (sperm do not transfer mt to the egg) ‒ almost the entire mt genome codes for protein subunits, most of which are related to the electron transfer or oxidative phosphorylation ‒ 13 genes code protein subunits, 2 rRNA genes, 22 tRNAs ‒  mitochondria must import most of their proteins from the cytosol outer mitochondrial membrane has high protein content: ‒ porins (transmembrane proteins) span outer mt membrane channels for water-soluble molecules up to ~1.5nm in size make outer membrane more permeable to small molecules, incl. proteins ‒ proteins that help synthesize mt lipids or aid in fatty acid metabolism ‒ receptors for proteins & polypeptides heading to the intermembranous space intermembranous (intermembrane) space ‒ space between inner & outer membranes ‒ contents aer similar to cytosol because of porin channels in outer mt membrane ‒ contains specialized enzymes that use ATP generated on the inner membrane one of these is cytochrome c  important in apoptosis ‒ mt decide whether cell lives/dies in times of stress ‒ release of cytochrome c into cytoplasm triggers proteolytic enzymes inner mitochondrial membrane is 75% protein!  extremely active: ‒ large protein complexes involved in: ‒ ATP synthesis (ATP synthases) ‒ forming the electron transport chain & moving electrons ‒ proteins involved in transferring the substrates and products of oxidative phosphorylation in/out of mitochondrial matrix ‒ some proteins are contact points between the inner & outer membranes ‒ regulate transport of proteins & small molecules in & out of matrix space ‒ macromolecules must be tagged with a marker to go through ‒ the inner membrane is nearly impermeable to ions, electrons, & protons high concentration of the phospholipid cardiolipin ‒ phospholipid with 4 fatty acid chains (vs 2) o heads of these project into the matrix o allows inner mitochondrial membrane to make hairpin turns  pack more cristae into mitochondrion o decreases the permeability of the inner mitochondrial membrane to charged particles (electrons)  reduces proton leak  ‒ proton leak (mitochondrial uncoupling) = protons re-enter mitochondrial matrix without contributing to ATP synthesis  this occurs independent of ATP synthase & is a decoupling of oxidation & phosphorylation  unharnessed potential energy of the proton electrochemical gradient is released as heat ‒ in most cells, proton leak is problematic (wastes energy, creates ROS) ‒ in brown fat cells, proton leak is intentional & mediated by an uncoupling protein (type of proton channel protein) called thermogenin  proton leak drives non-shivering thermogenesis ‒ cristae = infoldings of inner mt membrane increase surface area for proteins involved in ATP synthesis ‒ # and length of cristae increases as ATP demand increases most cells: cristae are lamellar (flat, shelf-like, & parallel to each other) steroid-synthesizing cells: cristae are tubular & randomly oriented matrix space is filled by matrix (mitochondrial matrix) ‒ matrix is viscous & gel-like because it is ~50% protein all but one of the enzymes involved in oxidation of pyruvate & fatty acids (i.e., the citric acid cycle enzymes) are located in the mitochondrial matrix ‒ mt genome (circular) also located in matrix ‒ mt ribosomes also located in matrix ‒ matrix contains electron dense matrix granules made of phospholipoprotein bind Ca2+  control mt calcium concentration D) Staining & EM LM: impart spots or striations of more intense acidophilia to cytoplasm ‒ individual mitochondria too small to see  striations/spots where they cluster ‒ can localize with histochemical stains or IHC targeted at mt-specific enzymes TEM ‒ double membrane: 2 bilayers (high mag) or 2 electron dense lines (lower mag) inner membrane curves inward to form cristae cristae may be shelf-like & parallel, or tubular at random angles (see above) ‒ matrix granules are very electron dense & larger than ribosomes IV) Energy Storage Forms: Glycogen & Lipid Droplets A) Glycogen glycogen is the most common storage form of glucose ‒ especially abundant in liver & muscle cells enzymes perform glycogenolysis on demand as needed ‒ degrades glycogen into glucose LM: glycogen granules: small & grainy not visible in H&E  in cells with large numbers of glycogen granules, there might be nonstaining regions of the cytoplasm (“glycogen ghosts”) PAS-positive (PAS, PAS + hematoxylin as counterstain)  EM: glycogen rosettes: clusters of electron-dense particles (glycogen molecules) rosettes vary in size, shape, & arrangement of electron-dense particles individual particles are smaller & more angular than ribosomes; never in spirals B) Lipid Droplets lipid droplets (fat droplets) = most common storage form of triglycerides very efficient energy reserves – 2x more ATP is derived from 1g of fat vs 1g of glycogen; also lipids do not swell with water (or take on water weight) may be stored in specialized cells (fat cells = adipocytes), or else in small droplets in other cell types (usually temporary; more common/persistent in liver & adrenal gland) LM: most solvents used during tissue preparation remove triglycerides, leaving empty spaces  frozen sections required does not stain in H&E (large white spaces) special stains: osmium stains TEM: spherical, homogenous droplets (round in 2D) of intermediate electron density hydrophobic  spontaneously fuse into large droplets “membrane” is 1 monolayer with fatty acid tails facing inwards (not a bilayer) Example Open-Ended Study Questions Study questions illustrate how each objective might be studied at appropriate detail for the exam. 1. Relate the structure of the smooth endoplasmic reticulum (SER) to its functions. 1a. How would you distinguish the SER from RER and the Golgi in a TEM image? Things to consider: position in cell, shape of cisternae, presence/absence of ribosomes, association with vesicles, staining patterns in H&E. 1c. Which membrane lipids are synthesized by the SER? Which of these lipids are eventually modified (glycosylated) by the Golgi? 1d. What is the role of flippase & floppase proteins in phospholipid synthesis? How do flippases & floppases contribute to the asymmetry in the types of phospholipids present in each monolayer of the SER membrane? 1e. Describe how newly-formed phospholipids are transferred from the SER to the following structures: mitochondria, Golgi, RER, cell membrane, lysosome, nucleus. 1f. Describe the LM staining patterns of a cell actively synthesizing large amounts of steroid hormones. Include information on the cytoplasm, SER, Golgi, secretory vesicles, and nucleus. Which of these structures would be most prominent in TEM images of steroid- secreting cells? 1g. In addition to lipid synthesis, what are the other main functions of SER? 2. Relate the structure of the mitochondrion to its functions. 2a. Compare and contrast the double membrane structure of the nucleus and mitochondria. Things to consider: shape of both membranes, role & structure of channel proteins, protein content of both membranes, structures visible in the inner compartment, relationship to other organelles. 2b. What are cristae and what is their function? Which membrane proteins (or protein complexes) are found on the matrix side of the inner mitochondrial membrane? Which reactions are those proteins involved with? 2c. What is cardiolipin? Where is it synthesized? How does it differ structurally from other membrane lipids, and what is its function? 2d. How does the shape of cristae differ in steroid-secreting cells vs most other cells? How does the number of cristae reflect the level of metabolic activity in a cell? 2e. What functions do mitochondria share with the SER? 3. Describe how energy is stored in glycogen granules & lipid droplets. 3a. Why do cells store glycogen and fat/lipid droplets? What is the advantage of storing energy as a lipid over glycogen? 3b. Both lipids and glycogen granules fail to stain in H&E. Describe why each substances is unstained in H&E. Which special stains would you use to show the presence of each? 3c. How would you distinguish glycogen granules from secretory vesicles in LM and TEM? 3d. How would you distinguish glycogen granules from free ribosomes/polyribosomes in TEM? 4. Describe examples of clinical significance relating to the SER, mitochondria, glycogen granules, & lipid droplets. 4a. Explain how SER is relevant to glycogen storage diseases. What would you expect to see in a TEM image of the cells of a patient who had a glycogen storage disease? What stain(s) for LM would be useful in diagnosing a patient with a glycogen storage disease? 4b. What is tolerance? What role does SER play in developing tolerance to alcohol & barbiturates? 4c. What is proton leak, and why is it problematic in most cells? 4d. Describe the role of mitochondria in initiating apoptosis..  FS1 29 / MSK 10 – Digestion, Recycling, & Waste Disposal – Fall 2024 Dr. Sarah Werning – [email protected] Lab 06 is associated with this lecture (in part) Recommended Readings GTH – Ch 2 (Cytoplasm): Endocytosis, Endosomes, and Lysosomes (skip Table 2.2); Peroxisomes; Proteasomes; Pigments; Figs 2.27, 2.28; Ch 9 (Nervous Tissue): section on Inclusions only; Fig 9.5 GCA – Ch 1 (The Cell): Endosomes, Lysosomes, Peroxisomes, Proteasomes, Inclusions; Ch 7 (Nervous Tissue): Plate 7-4 (Sympathetic Ganglia, Sensory Ganglia  Fig 2 only) W – Ch 1 (Cell Structure and Function): Figs 1.13, 1.24, 1.25; Ch 3 (Blood, Haematopoiesis, & Bone Marrow): Fig 3.10; Ch 6 (Muscle): Fig 6.23; Ch 7 (Nervous Tissues): Fig 7.21 BRS – Ch 1 (Cell): sections VII.A.8, 9, 10 (Lysosomes, Peroxisomes, Proteasomes); VIII.C (Intracellular Digestion); Fig 1.22 All plates and figures listed above, plus any figures from Lectures 10, 16 & 17 related to buildup and/or inclusions are helpful for Lab 6. Lecture Objectives See last 2 pages for example open-ended study questions for each lecture objective. After reviewing this lecture, the learner should be able to: 1. Relate the structure of endosomes & lysosomes to their functions. 2. Relate the structure of peroxisomes to their functions. 3. Relate the structure of proteasomes to their functions. 4. Describe how the cell uses endosomes, lysosomes, peroxisomes, and proteasomes to regulate the breakdown & buildup of macromolecules within the cytoplasm. Lecture Outline I) Intracellular Digestion A) Definition & Need breaking down biological macromolecules into basic components (within cytoplasm) several reasons cells need to do this, including: obtain nutrients they lack or can’t make  recycling: some molecules are in near-constant demand (e.g. amino acids) destroy old or malformed structures (worn out organelles, misfolded proteins) destroy foreign cells & pathogens chemicals & processes that break down macromolecules can destroy the cell itself require specific chemical environments for optimum performance ‒ in cytosol, they often don’t function (e.g., wrong pH) ‒ OR they break down the cell (the cell is made of macromolecules) organelles that digest have a specific chemical ID tag associated with them chemical “shipping labels” that direct a vesicle to the correct address receptors on the organelle won’t accept delivery without the correct label ‒ prevents accidental destruction from a misdelivered package B) Types of Intracellular Digestion described based on where the material is coming from each has a specific type of vesicle associated with it newly formed vesicle is proportional to the volume of the ingested material autophagy: intracellular digestion of components of the cell itself vesicle: autosome heterophagy: intracellular digestion of endocytosed materials (from outside cell) phagocytosis – “cell eating” – volume of material is large, so vesicle is large ‒ foreign cells, ECM fibers ‒ vesicle: phagosome pinocytosis – “cell drinking” – volume is smaller, so vesicle is smaller ‒ usually bringing in specific molecules (need receptor) ⇒ maintains cell size/shape: vesicles are constantly merging with cell membrane (e.g. secretion)  need to return excess membrane ‒ or: to remove excess cell membrane & return it for recycling ‒ vesicle: = pinocytotic vesicle or coated vesicle II) Endosomes & Lysosomes A) Overview maturation series of membrane-bound organelles with acidic lumen contents: ‒ early endosome  late endosome  lysosome ‒ these organelles ingest, sequester, or degrade materials that enter via endocytosis acidic lumen contents: acid hydrolases membranes promote low pH: have a H+ pump (proton pump) ‒ regulated by membrane-bound H+-ATPases ‒ lumen pH progressively lowers as organelles mature B) Early Endosomes irregular-shaped vesicle, lies closer to cell membranes lumen pH 22 C