Protein Distribution and Transport (Unit 5) PDF

Unit 5. Protein distribution and transport: ribosomes, endoplasmic reticulum, Golgi apparatus, and lysosomes. SECTION II: CELL STRUCTURE AND FUNCTION. INDEX 5.1. Protein synthesis, processing and regulation 5.2. Endoplasmic reticulum 5.3. Golgi apparatus 5.4. Vesicle transport mechanism 5.5. Lysoso...

Unit 5. Protein distribution and transport: ribosomes, endoplasmic reticulum, Golgi apparatus, and lysosomes. SECTION II: CELL STRUCTURE AND FUNCTION. INDEX 5.1. Protein synthesis, processing and regulation 5.2. Endoplasmic reticulum 5.3. Golgi apparatus 5.4. Vesicle transport mechanism 5.5. Lysosomes  Proteins carry out the functions determined by the information encoded in genomic DNA. Protein synthesis is the final stage of gene expression. The translation of the mRNA is only the first step in the constitution of a functional protein. Necessary folding and processing. Gene expression is also regulated at the translational level. Furthermore, different mechanisms control the activity of proteins and their quantity (differential degradation).  Eukaryotic cells have membrane-bound organelles in the cytoplasm. Protein transport between different organelles is a complex process.  General scheme of protein distribution in mammals Proteins synthesized on free ribosomes remain in the cytosol or are transported to the nucleus, mitochondria, chloroplasts, or peroxisomes. The proteins destined for the endoplasmic reticulum, Golgi apparatus, lysosomes, membranes and to be secreted, are synthesized in ribosomes bound to the ER membrane. In the ER, the folding and processing of these proteins takes place. Golgi From the ER, these proteins are transported in vesicles to the GA, where they are further processed and distributed. Peroxisome membrane Nuclear membrane 5.1. Protein synthesis, processing and regulation ❖ mRNA translation ❖ Protein folding and processing ❖ Regulation of protein function ❖ Protein degradation ❖ mRNA translation Transfer RNA Ribosomes Organization of mRNAs and initiation of translation Translation process Regulation of translation - Proteins are synthesized from a mRNA template. - The mRNAs are read in the 5’ to 3’ direction. - Polypeptide chains are synthesized from the amino terminus to the carboxyl terminus. - Each aa is encoded by 3 bases (a codon) in the mRNA [almost universal character of the genetic code]. - Translation takes place in ribosomes (rRNA + proteins). - The tRNAs are the adapters between the mRNA and the aa incorporated into the protein.  Transfer RNA They all have a similar structure: - 70-80 nt in length - cloverleaf shape due to the complementarity of bases between regions of the molecule - L-shaped folding - CCA sequence at the 3 'end. The aa are covalently attached to the ribose of the terminal adenosine. - Anticodon loop: it binds to the appropriate codon by 3-base complementarity. A) open model in the shape of a clover leaf B) folded form of the molecule C) three-dimensional model Aminoacyl tRNA synthetases: Very selective enzymes that attach the appropriate amino acid onto its corresponding tRNA. The mechanism can be summarized in the following reaction series: Amino Acid + ATP → Aminoacyl-AMP + PPi Aminoacyl-AMP + tRNA → Aminoacyl-tRNA + AMP Genetic code 64 possible codons 61 encode aa 3 are stop codons Most aa are encoded by more than one codon, there is more than 1 tRNA for the same aa Some tRNAs recognize more than one codon: nonstandard base pairing (wobble) Nonstandard codon–anticodon base pairing. This allows G to pair with U, and inosine (I) to pair with U, C, or A. (Guanosine is modified to inosine in the anticodons of some tRNAs.) ❖ Ribosomes rRNA is currently believed to catalyze the formation of the peptide bond. - Discovery of catalytic activity of other RNAs. - rRNA absolutely necessary for the in vitro assembly of functional ribosomes. In contrast, the lack of ribosomal proteins causes a decrease but not a complete loss of functionality. - First high resolution structural analysis (year 2000) revealed that ribosomal proteins were markedly absent from the peptide bond formation site.  mRNAs have noncoding untranslated regions (UTRs) at the ends. Organization of mRNAs and Most eukaryote mRNAs are mono-cistronic, encoding a single initiation of protein. translation Prokaryotic mRNAs are often poly-cistronic, encoding multiple proteins, each of which is translated from an independent start site. AUG codon: Start of translation with aa methionine (in most bacteria it is N-formylmethionine) The signals that identify initiation codons are different in prokaryotic and eukaryotic cells. Initiation codons in bacterial mRNAs are preceded by a Shine-Dalgarno sequence, that aligns the mRNA on the ribosome. They can initiate translation at the 5′ end of an mRNA and at internal initiation sites of polycistronic mRNAs. Eukaryotic mRNAs are recognized by the 7-methylguanosine cap at the 5′ end. The ribosomes then scan downstream of this cap until they encounter the initiation codon.  Translation process Many non-ribosomal proteins are also required for various stages of translation.  Initiation of translation in bacteria 1. 30S ribosomal subunit 2. The mRNA + initiator N- binds IF1 and IF3 formylmethionyl (fMet) tRNA + IF2 (bound to GTP) join the complex. 3. tRNA binds to the start 4. 50S subunit is associated codon and IF1 and IF3 are with the complex, which released induces the hydrolysis of GTP bound to IF2 and the release of An initiation complex is this factor. formed prepared to catalyze the formation of a peptide bond during elongation. Initiation of translation in eukaryotic cells (part 1) 1. eIF1, eIF1A and eIF3 bind to the 40S subunit 2. eIF2, bound to GTP, binds to 3. A pre-initiation complex is the initiator methionyl tRNA formed by associating all of this and eIF5 4. The cap at the 5 'end of the mRNA is recognized by eIF-4E, which forms a 5. These factors direct the complex with eIF-4A and mRNA towards the 40S eIF-4G. eIF-4A also binds to subunit through interaction eiF-4B and eIF-4G also between eIF-4G and eIF-3 binds to poly-A binding protein (PABP). Initiation of translation in eukaryotic cells (part 2) 6. The 40S subunit bound to methionyl tRNA and the eIF check the mRNA until an AUG start codon is identified. Displacement requires energy. 7. When AUG is recognized, eIF- 5 causes hydrolysis of the GTP bound to eIF-2 and the eIFs are released. eIF5B, initially bound to GTP, facilitates the binding of the 60 S subunit.  1. The initiator methionyl Elongation tRNA binds to the P site. Ribosomes have three binding sites: P (peptidyl) 2. An elongation factor (EF-Tu in A (aminoacyl) prokaryotes, eEF1α in eukaryotes) E (exit) complexed to GTP brings the aminoacyl tRNA to the ribosome. 5. The ribosome then moves 3 nucleotides along the mRNA, positioning the next codon in the A site. 3. The next aminoacyl tRNA binds to This step translocates the peptidyl tRNA from A to the A site by pairing with the second P, and the uncharged tRNA from P to E. codon of the mRNA. Hydrolysis of GTP and release of the EF. 6. When the next aminoacyl tRNA 4. The peptide bond is then binds to site A, formed, catalyzed by the large free tRNA from ribosomal subunit. site E is released. The initiator tRNA (uncharged) 5b. Translocation requires EF-G in prokaryotes and is now at the P site. eEF2 in eukaryotes, and is coupled to GTP hydrolysis. https://www.youtube.com/watch?v=3proluePeVs Termination Elongation continues until a stop codon (UAA, UAG, or UGA) is translocated into the A site. Release factors recognize these signals and terminate protein synthesis.  Once a ribosome has moved away from the initiation site, The mRNAs are normally translated another can bind to the mRNA and begin synthesis. by a series of ribosomes, separated A group of ribosomes bound to an mRNA molecule is called by about 100-200 nt. a polyribosome, or polysome.  Regulation of translation - Transcription is the main level of control of gene expression. - Regulation of the translation of specific mRNAs is also important in modulating gene expression. - Translation repressor proteins - noncoding micro-RNAs - Translation can also be globally repressed in situations of cellular stress, such as starvation, depletion of growth factors, or DNA damage. - Modulation of the activity of eIF-2 and eIF4E. Translational repressor bound to the 3 'untranslated regions. Translational repressors can bind to regulatory sequences of the 3’ untranslated region (UTR) and inhibit translation by binding to the initiation factor eIF4E, attached to the 5' cap. This interferes with translation by blocking the binding of eIF4E to eIF4G. Regulation of ferritin translation (a protein that stores iron) by repressor proteins: When iron is absent, iron regulatory protein (IRP) binds to the iron response element (IRE) in the 5′ UTR, blocking translation. Translation can also be regulated by modification of initiation factors. This results in global effects on overall translational activity rather than on translation of specific mRNAs. Phosphorylation of eIF2 and eIF2B by regulatory protein kinases blocks the exchange of bound GDP for GTP, inhibiting initiation of translation.  Regulation of eIF4E. In the absence of growth factors, eIF4E-binding proteins (4E-BP) bind to this factor and prevent its association with eIF4G, inhibiting translation. Stimulation by growth factors induces phosphorylation of 4E-BP, which dissociates from eIF4E and allows translation to begin. ❖ Protein Folding and Processing Chaperones and protein folding Diseases due to protein folding defects Enzymes that catalyze protein folding Proteolysis Glycosylation Lipid binding  To be active, polypeptides must fold into characteristic three- dimensional conformations. In many cases, several polypeptide chains aggregate to form a functional complex. Many proteins undergo additional modifications: cleavage, covalent union of carbohydrates and lipids, necessary for the correct cellular function and localization.  Chaperones and protein folding Chaperones: proteins that facilitate the folding of other proteins or assembly processes of complexes involving proteins. They do not provide additional information for the folding of proteins in their three- dimensional conformation; it is determined exclusively by its amino acid sequence (interaction between the side chains). They bind to and stabilize unfolded or partially folded polypeptide, preventing aberrant folding or aggregation. There are many types: chaperones that bind to nascent polypeptide chains chaperones that stabilize partially folded polypeptides in their transport to cellular organelles chaperones involved in the assembly of multiple polypeptide chains chaperones that participate in the assembly of macromolecular structures (eg. nucleoplasmin) This type of chaperones prevent misfolding or aggregation of the amino terminal portion of the polypeptide before the synthesis of the polypeptide is complete. This type of interaction is important in proteins where the carboxyl terminal end (synthesized last) is necessary for the folding of the amino terminal end. The bound chaperone stabilizes the amino terminal portion in an extended conformation until the polypeptide is completely synthesized. Thus, the entire polypeptide chain can be correctly folded. Role of chaperones during protein transport. A partially folded polypeptide is transported from the cytosol to the mitochondria. Cytosolic chaperones stabilize the extended conformation. Mitochondrial chaperones facilitate the transport and subsequent folding of the polypeptide chain within the organelle. Sequential actions of chaperones. Hsp70 proteins stabilize polypeptide chains during translation. The polypeptide is then transferred to a chaperonin, where folding takes place. Chaperonins consist of subunits arranged in two stacked rings to form a double-chambered structure. This isolates the protein from the cytosol and other unfolded proteins.  Diseases due to protein folding defects - Aggregation of misfolded proteins. - Reduction of functional protein levels. Eg. cystic fibrosis. Mutation for the CFTR protein. https://www.youtube.com/watch?v=6IbP1ASGv9w  Enzymes that catalyze protein folding - Protein disulfide isomerase (PDI) It catalyzes the formation of disulfide bonds between cysteine residues, which are very important in the stability of the folded structure of many secreted and membrane proteins. PDI is abundant in the ER, where an oxidizing environment allows (S—S) linkages.  Proteolysis Important stage in the maturation of many proteins. - Removal of the initial methionine from the amino terminus of many polypeptides. It occurs at the beginning of the translation. - New chemical groups are often added to the amino terminus, such as acetyl or fatty acid chains. - Elimination of the amino-terminal signal sequence in secretory proteins or membrane proteins in eukaryotic cells (see section 5.2). - Formation of functionally active proteins by cleavage of larger inactive precursors. Eg. insulin, digestive enzymes, proteins from many animal viruses, such as HIV. Insulin is synthesized as a precursor polypeptide that goes through two cleavages to produce the mature insulin.  Glycosylation Addition of carbohydrate chains to proteins to form glycoproteins. More frequent in eukaryotic cells. They are normally secreted or located on the cell surface. Functions: - Protein folding in the ER - Targeting proteins to their intracellular compartment - Recognition in intercellular interactions Types of glycoproteins depend on the binding site of the carbohydrate chain: - N-linked glycoproteins: the carbohydrate is attached to the nitrogen atom in the side chain of asparagine. - O-linked glycoproteins: the carbohydrate is attached to the oxygen atom in the side chain of serine or threonine.  Lipid binding Binding of lipid molecules to the polypeptide chain. They frequently mark and anchor these proteins to the plasma membrane, being the lipid, by its hydrophobic nature, the one that is inserted into the membrane. ❖ Regulation of Protein Function Regulation by small molecules Phosphorylation and other modifications Protein-protein interactions  Regulation by small molecules Most enzymes are regulated by changes in their conformation that cause modifications in their catalytic activity. These changes are often produced by the non- covalent union of small molecules (aa, nt.). Allosteric regulation: a regulatory molecule binds to an enzyme site other than the active center, modifying the protein's conformation and affecting its activity. - Retrohinibition of metabolic pathways - Transcription factors - Regulation of translation factors such as eEF-1α by binding of GTP or GDP  Phosphorylation and other modifications Phosphorylation is the most common and best-studied type of covalent modification that regulates protein activity in response to environmental signals. Protein kinases transfer phosphate groups from ATP to the hydroxyl groups of side chains of serine, threonine, or tyrosine. Protein kinases are often components of signal transduction pathways. Phosphorylation is reversed by protein phosphatases, which catalyze hydrolysis of phosphorylated amino acids. Other modifications by covalent bonding: acetylations (addition of COCH3 groups) of lysine residues methylations (addition of CH3 groups) of lysine and arginine glycosylation of serine and threonine nitrosylation (addition of NO groups) of cysteine residues peptide addition ubiquitylation (addition of ubiquitin (76aa)) sumoylation (addition of SUMO proteins)  Protein-protein interactions Many proteins consist of multiple subunits; interactions between them can regulate protein activity. Example: cAMP-dependent protein kinase has two regulatory and two catalytic subunits in the inactive form. cAMP binds to the regulatory subunits, which induces conformational change and dissociation of the complex. The free catalytic subunits are then enzymatically active protein kinases. ❖ Protein degradation The amount of proteins in the cell is regulated not only by their rate of synthesis but also by their rate of degradation. The half-life of proteins is highly variable, from a few minutes to several days. Regulatory proteins, such as TFs, are rapidly degraded. Other proteins break down in response to specific signals. Defective or damaged proteins are also degraded. Ubiquitin-proteasome pathway Lysosomal proteolysis  The ubiquitin-proteasome pathway The main route of selective protein degradation in eukaryotes involves ubiquitin as a protein marker. Polyubiquitinated proteins are recognized and degraded by a large complex with multiple subunits and protease activity, the proteasome.  Protein degradation can also take place in Lysosomal proteolysis lysosomes—membrane-enclosed organelles that contain digestive enzymes, including proteases. Protein capture in autophagosomes is not a selective process. Lysosomes digest extracellular proteins taken up by endocytosis, and take part in turnover of organelles and proteins. Proteins move into lysosomes by autophagy: vesicles (autophagosomes) enclose small areas of cytoplasm or organelles and then fuse with lysosomes. Autophagy is activated in nutrient starvation, allowing cells to degrade nonessential proteins and organelles and reutilize the components. 5.2. Endoplasmic reticulum Membrane-surrounded network of tubules and sacs extending from the nuclear membrane to the entire cytoplasm It is surrounded by a continuous membrane. Its membrane constitutes 50% of all cell membranes and its lumen (or cistern space) 10% of the cell volume. ▪ Rough ER (RER): associated with ribosomes on its outer (cytosolic) face and involved in protein synthesis and processing. ▪ Smooth ER (SER): does not associate with ribosomes and is involved in lipid metabolism.  RER and protein secretion. Label-chasing experiment [George Palade (1960s)] Pancreatic acinar cells, which secrete most of their de novo synthesized proteins in the digestive tract, were labeled with radioactive amino acids to study the intracellular pathway used by secreted proteins. After a short incubation period with radioactive amino acids (3-minute label), autoradiography revealed that the de novo synthesized proteins were localized in the rough ER. After a subsequent incubation with non-radioactive amino acids (chasing), it was observed that the proteins had moved from the ER to the Golgi apparatus and then, inside secretory vesicles, to the plasma membrane and to the outside of the cell.  The proteins synthesized in the RER are translocated directly into the ER through the translocon. They can be retained within it or be transported to the membranes of the nucleus or peroxisomes, or to the Golgi apparatus, and from this, to Golgi endosomes, lysosomes, plasma membrane or be secreted through the secretory vesicles. Peroxisome membrane Nuclear membrane  ❖ Labeling of proteins to target the ER Proteins can be translocated to the ER during their synthesis on membrane-bound ribosomes (cotranslational translocation) or once translation has been completed on free ribosomes in the cytoplasm (post-translational translocation). In mammalian cells, most proteins enter the ER in a cotranslational manner, while in yeast both the cotranslational and posttranslational pathways are used. The first step in the cotranslational pathway is the association of the ribosome-mRNA complex with the ER. The 'tag' that determines that the ribosome binds to the ER membrane is contained in the primary sequence of aa. of the polypeptide chain being synthesized, it is not due to intrinsic ribosome properties. It is the signal sequence. Free and membrane-bound ribosomes are functionally indistinguishable, and all protein synthesis begins on ribosomes that are free in the cytosol.  This signal sequence contains between 15-40 aa, including a strip of 7-12 hydrophobic amino acids, usually at the amino terminal end of the polypeptide chain, preceded by basic aa like Arginine (Arg). Growth hormone signal sequence  Cotranslational translocation of secretion proteins to the ER 1. As they exit the ribosome, the signal sequences are recognized and attached to a signal recognition particle (SRP). 2. SRP stops translation and accompanies the complex to the ER membrane, where it binds to the SRP receptor. 3. The SRP is released, and the ribosome binds to the translocon. The insertion of the signal opens the translocon. In yeast and mammalian cells, translocons are complexes of three transmembrane proteins called Sec61. 4. Translation resumes and the signal sequence is cleaved by a signal peptidase. 5. Continuation of translation drives the translocation of the growing polypeptide chain across the membrane. 6. The polypeptide, once completed, is released into the lumen of the ER. https://www.youtube.com/watch?v=FRph3TGkIAE  Post-translational translocation Proteins are translated by free cytosolic ribosomes and then targeted to the ER (this occurs in many yeast proteins). The post-translational incorporation does not require SRP. Their signal sequences are recognized by the Sec62/63 complex associated with the translocon in the ER membrane. Hsp70 chaperones are required to maintain the polypeptide chains in their primary conformation so that they can penetrate the translocon. Other Hsp70 chaperones within the ER (called BiP), associated with Sec63, are necessary to allow the polypeptide chain to cross the channel into the ER. Many BiP molecules bind to the polypeptide chain as they undergo translocation to the ER, preventing them from sliding backward and effectively propelling them through the channel. The release of BiP molecules is linked to the hydrolysis of ATP. Post-translational translocation is activated by BiP, and cotranslational translocation is directly driven by the process of protein synthesis.  ❖ Insertion of proteins into the ER membrane Proteins destined to be secreted or reside in the ER lumen, Golgi apparatus, or lysosomes are translocated and released into the ER lumen. Proteins destined to be incorporated into the plasma membrane or ER membrane, Golgi, or lysosomes are initially inserted into the ER membrane rather than being released into the ER lumen. From the ER membrane they continue to their final destination by the same route as secreted proteins [ER - Golgi - Plasma membrane or lysosomes], but are transported along this route as components of the membrane, rather than as soluble proteins..  Integral membrane proteins are embedded in the membrane by hydrophobic regions that cross the lipid membrane. The regions of these proteins that cross the lipid bilayer are usually α-helix regions made up of 20 to 25 hydrophobic aa. The formation of an α-helix: maximizes hydrogen bonds between peptide bonds hydrophobic side chains of amino acids interact with fatty acid tails of phospholipids The various integral membrane proteins differ in how they are inserted Some integral proteins cross the membrane only once, others have multiple regions that cross the membrane. Some proteins are oriented with their carboxy-terminal end on the cytosolic side; others have their amino-terminal end exposed on the cytosolic side.  Most of the membrane proteins are inserted into the membrane by the cotranslational pathway. The orientation of the inserted protein membranes is established as the growing polypeptide chains translocate into the ER. The ER lumen is topologically equivalent to the outside of the cell, so the domains of the plasma membrane proteins that are exposed on the cell surface correspond to the regions of the polypeptide chain that are translocated inside the ER.  Many of the proteins are inserted directly into the ER membrane by internal transmembrane sequences that are recognized and translocated by SRP, but are not cleaved by signal peptidase. In these cases there is no amino-terminal signal recognized by SRP, but rather recognizes the internal (transmembrane) signal sequence. The helix chain exits the translocon laterally and binds the protein to the ER membrane. The hydrophobic transmembrane sequence indicate a change in the translocon, thereby causing the hydrophobic transmembrane domain of the protein to exit the translocon into the lipid bilayer. Depending on the transmembrane signal sequences, proteins inserted into the membrane by this mechanism may have an amino or carboxyl end exposed in the cytosol. Thus, the orientation of the inserted protein membranes is established as the growing polypeptide chains translocate into the ER. A) The internal transmembrane sequence directs the insertion of the polypeptide so that its amino (N) end is exposed on the cytosolic side. The transmembrane sequence exits the translocon to fix the protein in the lipid bilayer and the rest of the polypeptide chain is translocated in the endoplasmic reticulum as translation proceeds. N B) The internal transmembrane sequence directs the insertion of the polypeptide so that its carboxyl terminus (C) is exposed on the cytosolic side. Other internal transmembrane sequences are oriented to direct the transfer of the amino-terminal part of the polypeptide across the membrane. Continuous translation produces a protein with its N end in the lumen and the C end in the cytosol. Insertion of a protein that crosses the membrane several times. Proteins with a transmembrane sequence at their carboxy-terminal end are inserted into the ER membrane by an alternative post-translational pathway These proteins cannot be recognized by SRP, because their transmembrane domain does not emerge from the ribosome until translation is complete and the entire polypeptide chain is released from the ribosome. Transmembrane domains are recognized by a targeting factor called TRC40 or GET3, once translation is complete. TRC40 escorts the transmembrane protein to the ER membrane, where it inserts through the GET1-GET2 receptor. Post-translational insertion of a protein with a C-terminal transmembrane sequence.  ❖ Protein folding and processing in the ER In the case of secretory proteins, many of these processes occur during translocation across the ER membrane or into the ER lumen. One of these processes is the cleavage of the signal sequence peptide as the polypeptide chain translocates across the ER membrane. The RE is also the place of: ✓ protein folding ✓ assembly of proteins from various subunits (quaternary structure) ✓ disulfide bond formation ✓ N-glycosylation ✓ addition of glycolipid anchors to some proteins of the plasma membrane The main role of ER lumen proteins is to catalyze the folding and assembly of the newly translocated polypeptides.  ❖ Quality control in the ER Many proteins synthesized in the ER are rapidly degraded, mainly because they do not fold correctly. These misfolded proteins are removed from the ER thanks to a process known as ER- associated degradation (ERAD), by which misfolded proteins are identified and leave the ER to the cytosol, where they are degraded by the ubiquitin-proteasome system. The chaperones and protein-processing enzymes of the ER lumen act as sensors for proteins with folding defects. A well characterized pathway of glycoprotein folding is related to the chaperones calnexin and calreticulin. Calnexin and calreticulin recognize partially processed oligosaccharides, from which two terminal glucose residues have been removed. Calnexin or calreticulin facilitate the folding of the glycoprotein into its correct conformation. The glycoprotein is released by the removal of a third terminal glucose residue from the oligosaccharide. This allows the recognition of the glycoprotein by a protein folding sensor that monitors whether the protein has reached a fully folded state (absence of exposed hydrophobic regions). If the folding is adequate, the protein continues towards the Golgi apparatus.  Otherwise, the folding sensor would add a glucose residue to the oligosaccharide, going into a new cycle with the clanexin or calreticulin for another attempt to correct the folding. If, after several cycles, the glycoprotein still does not fold correctly or shows an irreversible folding defect, it is directed to the ERAD pathway for degradation. The protein with the folding defect is recognized by an enzyme called EDEM1 that removes the mannose residues from the oligosaccharide. Removal of mannose prevents the folding defective glycoprotein from being returned to calnexin or calreticulin, and causes them to be transferred to a transmembrane complex with ubiquitin-ligase activity. They are then translocated to the cytosol where it is ubiquitylated and degraded in proteasomes. The amount of unfolded proteins in the ER is monitored in order to coordinate the ability to fold proteins in the ER with the physiological needs of the cell. This regulation is mediated by a signal pathway known as the Unfolded Protein Response (UPR) that is activated if too many unfolded proteins accumulate in the ER. Activation of the UPR leads to the expansion of the ER and the production of more chaperones to meet the demand for protein folding, as well as a transient reduction in the amount of de novo synthesized proteins that reach the ER. If these modifications are insufficient to cover the protein folding needs and an accumulation of unfolded proteins occurs in the ER, the cell would be doomed to programmed cell death, thus eliminating the cells of an organism that are incapable of folding. proteins correctly.  In mammals, the UPR is made up of three proteins: IRE1, ATF6 and PERK IRE1: results in the cleavage and activation of the mRNA encoding XBP1 on the cytosolic face of the ER. o XBP1 is a transcription factor that induces the expression of chaperone genes, lipid synthesis enzymes, and ERAD proteins. ATF6: is a transcription factor retained on the cytosolic face of the ER membrane. In cells subjected to protein stress with folding defects, ATF6 is transferred to the Golgi apparatus, where it is cleaved to release its active form to the cytosol. From there it goes to the nucleus where it will induce the expression of more genes that respond to unfolded proteins. PERK: protein kinase that phosphorylates and inhibits the translation initiation factor (eIF2). o This results in an overall reduction in protein synthesis and thus in a decrease in the load of misfolded proteins in the ER. o It also leads to preferential translation of certain mRNAs, leading to increased production of ATF4, a transcription factor that further contributes to the production of proteins involved in UPR.  ❖ Smooth ER and lipid synthesis The SER is the main site where lipids of eukaryotic cell membranes are synthesized. Since they are extremely hydrophobic, lipids are synthesized in association with existing cell membranes. Most lipids are synthesized in the membranes of the SER and are transported by vesicles or carrier proteins. The plasma membrane is made up of three types of lipids: phospholipids, glycolipids, and cholesterol. Phospholipids are the fundamental components of cell membranes, derived mostly from glycerol and are synthesized on the cytosolic side of the SER membrane from water- soluble cytosolic precursors. SYNTHESIS OF PHOSPHOLIPIDS 1. Fatty acids transfer from CoA transporters to Glycerol-3-phosphate, giving rise to phosphatidic acid. 2. Enzymes of the cytosolic side of the SER catalyze the addition of different polar head groups, giving rise to: phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. The synthesis of these phospholipids on the cytoplasmic side of the ER allows the hydrophobic chains to remain hidden in the membrane, while the membrane-bound enzymes catalyze their reactions with water-soluble precursors (such as CDP-choline) in the cytosol.  Translocation of phospholipids across the ER membrane Since phospholipids are synthesized on the cytosolic side of the SER membrane, they are only added to the middle of the lipid bilayer. They are subsequently translocated through the membrane by phospholipid flippases, giving rise to a uniform growth of the two halves of the phospholipid bilayer. There are different families of flippases, some of which are specific to specific phospholipids.  The ER is also the main site for the synthesis of other membrane lipids: cholesterol and ceramide. Ceramide is converted to glycolipids or sphingomyelin in the Golgi apparatus. SER is abundant in cells that are active in lipid metabolism. E.g. steroid hormones are synthesized in ER from cholesterol, so SER is especially abundant in steroid- producing cells, such as in testes and ovaries.  ❖ Export of proteins and lipids from the ER Protein and lipids are exported from the ER in vesicles that undergo budding from specialized regions of the ER called ER exit sites, or Lumenal ERESs. proteins The vesicles fuse to form the ER-Golgi intermediate compartment (ERGIC), from which the load is transported to the GA. Membrane proteins and lipids are transported in a similar way, maintaining their orientation from one organelle to another. Golgi-targeted ER luminal proteins are bound by membrane proteins that are selectively packaged into vesicles. Later they are released in the AG. Resident ER proteins destined to remain in the ER light are marked by KDEL (Lys-Asp-Glu-Leu) retrieval sequences at their carboxyl terminus. If these proteins are exported from the ER to the Golgi, they are recognized by a recycling receptor in ERGIC or the Golgi apparatus and selectively returned to the ER. 5.3. Golgi apparatus Many of the proteins synthesized and processed in the ER reach the Golgi Apparatus and are reprocessed and distributed to other destinations, if applicable: lysosomes, the plasma membrane or secretion. In the GA, glycolipids and sphingomyelin are synthesized. It is involved in processing a wide spectrum of cellular constituents that travel along the secretory pathway.  ❖ Golgi Organization The GA is composed of flattened bags, surrounded by a cis face membrane (cisterns) and associated vesicles GA is a polar organelle, both structurally and functional. Proteins from the ER enter through the convex cis face (entrance face), which is usually oriented towards the nucleus. They are then transported through the GA and exit through its concave exit face (trans face). As they pass through the GA, proteins are modified and distributed for transport to their final destinations in the cell. trans face  The GA is divided into four different functional regions: cis, medial, trans, and the trans-Golgi network. The different processing and distribution processes take place in an ordered sequence in the different compartments of the GA. The proteins of the ER-Golgi intermediate compartment enter the cis compartment of the GA, in which the modification reactions of proteins, lipids and polysaccharides begin. Later they cross the medial and trans compartments, in which they are subjected to further modifications, and finally migrate to the Golgi trans network, which acts as an organization and distribution center that directs molecular traffic towards the different destinations: endosomes, lysosomes, plasma membrane or cell exterior.  ❖ Protein glycosylation in the Golgi apparatus Protein processing in the GA involves extensive modification of the carbohydrate regions of glycoproteins The GA contains more tan 250 enzimas, which catalyze the addition of different sugars to the glycoproteins. These enzymes are placed in different compartments of the GS, so that carbohydrate processing takes place in order. Translocation in the ER. The oligosaccharide is transferred as a unit to the acceptor Asn residues in the consensus sequence Asn-X-Ser/Thr by an enzyme called Oligosaccharyl-transferase. Three glucose residues are eliminated while the protein is in the ER lumen. This protein undergoes additional modification in the GA. Processing of N-oligosaccharides in the Golgi The N-glycosaccharides of the glycoproteins transported from the ER are subsequently modified by an ordered sequence of reactions catalyzed by enzymes in different compartments in the Golgi. cis The different glycoproteins are modified in different ways during their passage through the GA, depending on the structure of the protein and the processing medial enzymes present in the Golgi complexes of each cell type. Consequently, proteins can leave the Golgi with different oligosaccharides attached to their N-terminal group. trans and trans-Golgi network Labeling and targeting of lysosomal proteins by phosphorylation of mannose residues The N-oligosaccharide processing of proteins recognition of “signal regions” destined to be incorporated into lysosomes differs from that of secreted proteins and the plasma membrane. Instead of the initial removal of mannoses, they are modified by phosphorylation of mannose. Due to this modification, these residues are not 1st: N-acetylglucosamine phosphate is added to removed during further processing. specific mannose residues from UDP-N acetylglucosamine. These mannose-6-phosphate residues are specifically recognized by a mannose-6-phosphate 2nd: The N-acetylglucosamine groups are receptor in the Golgi trans network, which directs eliminated giving rise to mannose-6-phosphate. the transport of these proteins to the lysosome.  Proteins can also be modified by adding carbohydrates to sequence-specific Ser and Thr residue side chains (O-glycosylation) Some O-linked oligosaccharides consist of only a few sugar residues, while others are long chains of many sugars. Proteoglycans, for example, which are secreted proteins that are important components of the extracellular matrix are an example of extended O-glycosylation. Its processing in the GA involves the addition of 100 or more carbohydrate chains to a polypeptide, where each chain is made up of up to 100 sugar residues that are modified by the addition of phosphate groups. Proteoglycan structure  ❖ Lipid and polysaccharide metabolism in the Golgi GA participates in lipid metabolism: Glycolipids and sphingomyelin are synthesized in GA from ceramide. glycolipids and sphingomyelin Sphingomyelin is the only non-glyceric phospholipid in cell membranes and is synthesized by the transfer of a phosphorylcholine group from phosphatidylcholine to ceramide. Alternatively, the addition of carbohydrates to the ceramide can give rise to different glycolipids. Sphingomyelin is synthesized on the luminal side of the Golgi, while glucose is added to ceramide on the cytosolic side. The glucosylceramide is turned over, and the additional sugars are incorporated into the luminal face of the Golgi. Glycolipids, as well as sphingomyelin, are not capable of translocating in the Golgi lipid bilayer, thus they are only found in the luminal half of the Golgi membrane. After vesicular transport, these glycoproteins are located on the outer face of the plasma membrane.  ❖ Distribution and export of proteins from the GA: secretory pathway Proteins are distributed in different types of transport vesicles, which bud from the trans Golgi region and carry their content to the appropriate cellular location. Some proteins are transported to the plasma membrane directly in vesicles; others through recycling endosomes as an intermediate compartment; and others are secreted through secretion granules* (regulated secretion, eg: hormones in endocrine cells, neurotransmitters in neurons, digestive enzymes in pancreatic acinar cells). Others target lysosomes. immature secretory Most of the resident Golgi proteins are transmembrane proteins that act in granule glycosylation and are retained within. * Proteins are distributed to the regulated secretory pathway in the Golgi trans Network where they are packaged into immature secretory granules. These will mature and expel their content to the outside in response to certain signals.  5.4. Vesicle transport mechanism Vesicle transport is a fundamental cellular activity Responsible for cell traffic between the various compartments surrounded by membrane. Vesicular transport is key to maintain the functional organization of the cell. Specificity in transport is based on the selective packaging of the selected cargo in vesicles, which recognize and fuse only to the appropriate target membrane. The molecular mechanisms that control vesicle packing, budding, distribution, and fusion is a major area of research in cell biology.  Selection of cargo, coat proteins and vesicular budding Most of the transport vesicles that carry proteins from the ER to the Golgi and the posterior compartments are coated with coat proteins and are called coated vesicles. The assembly of the coat proteins drives the budding of vesicles containing selected cargo proteins from the donor membrane. Vesicles travel along cytoskeletal filaments to their destinations, by the action of motor proteins. The coatings are removed on the target membranes, allowing the membranes to fuse, and the vesicles evacuate their luminal cargo and insert their membrane proteins into the target membrane.  Three families of vesicle coat proteins are known: clathrin, COPI and COPII (COP, from coat complex protein). COPII-coated vesicles are responsible for transporting proteins from the ER to the ER-Golgi intermediate compartment and the Golgi apparatus. The COPI-coated vesicles leave the ER-Golgi intermediate compartment or the GA by budding and carry their cargo in a retrograde direction to return the resident proteins to the anterior compartments of this pathway (they are distributed throughout the secretory system between ER and AG). Clathrin-coated vesicles are responsible for bidirectional transport between the Golgi trans network, endosomes, lysosomes, and the plasma membrane.  The fusion of a transport vesicle with its target involves two types of events 1. The transport vesicle must specifically recognize the correct target membrane. 2. The vesicle membrane and the target membrane must fuse, delivering the contents of the vesicle to the target organelle. The initial interaction between transport vesicles and specific target membranes is mediated by anchoring factors and small GTP-binding proteins called Rab (active state: Rab-GTP). Their binding provides the initial bridge between the target membranes and the vesicles. Complexes are then formed between transmembrane proteins called SNARE in the vesicle and target membranes, leading to fusion of the membranes. All SNARE proteins have a long central coiled α-helix domain. The interaction of these coiled structures between SNARES (vesicle-target) favors the fusion of the membranes.  More than 60 different Rab proteins have been identified that function in vesicle-specific transport. Different Rab proteins or combinations of Rab proteins mark different transport organelles and vesicles. 5.5. Lysosomes Lysosomes are membrane-bound organelles that contain a series of enzymes capable of degrading all kinds of biological polymers (proteins, nucleic acids, carbohydrates, and lipids). They function as the cell's digestive system, serving both to degrade material captured from outside the cell and to digest obsolete components of the cell itself. In their simplest form, lysosomes appear as dense spherical vacuoles, but they can vary in size and shape depending on the different materials they have captured.  Lysosomes represent morphologically diverse organelles defined by the common function of degrading intracellular material Transmission electron micrograph showing lysosomes and mitochondria in a mammalian cell. Arrows indicate the variety of lysosomes seen in this micrograph. Note the difference in sizes and shapes, determined by the differences in the materials captured to be digested.  Acid lysosomal hydrolases Lysosomes contain about 60 different degradative enzymes that can hydrolyze proteins, DNA, RNA, polysaccharides, and lipids. Mutations in the genes that code for these enzymes are responsible for more than 30 different human congenital diseases called lysosomal storage diseases, since the non- degraded material accumulates in the lysosomes of affected individuals (eg Gaucher disease). Most lysosomal enzymes are acid hydrolases, which are active at the acidic pH (approximately 5) inside the lysosomes, but not at the neutral pH (approximately 7.2) characteristic of the rest of the cytoplasm. The need for these lysosomal hydrolases to be acidic provides protection against uncontrolled digestion of cytosol contents. To maintain the internal acidic pH, H+ (protons) have to be actively incorporated (need of ATP) by a proton pump into the lysosome membrane. This active transport maintains the concentration of H+ 100 times higher in the lysosome than in the cytoplasm.  Endocytosis and lysosome formation One of the main functions of lysosomes is the digestion of material captured from outside the cells through endocytosis. Lysosomes are formed by the fusion of transport vesicles originating from the trans Golgi network, which carry lysosome proteins, with a late endosome, which contain molecules ingested by endocytosis from the plasma membrane. The formation of endosomes and lysosomes thus represents an intersection between the secretory pathway, through which lysosomal proteins are processed, and the endocytic pathway, through which extracellular molecules are ingested from the cell surface.  Animal cells have three types of endosomes: early, recycling, and late. Early endosomes receive vesicles formed by endocytosis in the plasma membrane. Material from the exterior of the cell is ingested into clathrin-coated endocytic vesicles, which originate by budding from the plasma membrane and subsequently fuse with early endosomes. The components of the membrane are recycled to the plasma membrane through recycling endosomes. Early endosomes mature into late endosomes, which are the precursors of lysosomes. Lysosomal acid hydrolases are transported to late endosomes from the trans-Golgi network. Lysosomal proteins are targeted to lysosomes by mannose-6-phosphate residues, which are recognized by mannose-6-phosphate receptors in the trans- Golgi network and packaged in clathrin-coated vesicles. Those receptors are later recycled.  Phagocytosis and autophagy Lysosomes also digest material from two other pathways: phagocytosis and autophagy. In phagocytosis, specialized cells, such as macrophages, ingest and degrade large particles (bacteria, cell debris, or aging cells that must be eliminated from the body). These particles are ingested into phagocytic vacuoles (phagosomes), which subsequently fuse with lysosomes (phagolysosomes), leading to the digestion of their content. Autophagy is a function of all cells and results in the gradual replacement of the cell's own components. First, a cytosolic membrane envelope forms around a cytoplasmic portion or an internal organelle (eg, a mitochondrion). The vesicle thus formed (autophagosome) fuses with a lysosome (phagolysosome) and its contents are digested. Autophagy plays an important role in many developmental processes, such as the metamorphosis of insects, which involves tissue remodeling and degradation of cellular components. Autophagy is activated when there is deprivation of nutrients, allowing the degradation of non-essential macromolecules and the reuse of essential components.  Gaucher’s disease Deficiency of the lysosomal enzyme glucocerebrosidase, which catalyzes the hydrolysis of glucocerebroside to glucose and ceramide It is the most common lysosomal storage disease. The accumulation of non-degraded products leads to an increase in the size and number of lysosomes, which produces cellular dysfunction and pathological consequences in the affected organs. The disease has three variants with different degrees of severity and that can affect the nervous system.