The Endomembrane System and Membrane Trafficking PDF

The Endomembrane System and Membrane Trafficking Amir Mhawi, DVM, PhD [email protected] Learning Objectives • Identify the steps of the secretory pathway and know which types of newly translated proteins enter the secretory pathway and which ones don’t. • Explain the modifications that occur to pro...

The Endomembrane System and Membrane Trafficking Amir Mhawi, DVM, PhD [email protected] Learning Objectives • Identify the steps of the secretory pathway and know which types of newly translated proteins enter the secretory pathway and which ones don’t. • Explain the modifications that occur to proteins and lipids as they pass through the rER and Golgi (N- and O-linked glycosylation, modification of N-linked sugars in the Golgi, formation of glycolipids, addition of sialic acid). • Explain the functions of SRP, the translocon, ER signal sequence, SNARES, Rabs, dynamin, HSP70, BiP, the M6P signal, clathrin, adaptins and an “eat me signal”. • Describe the role of the mannose-6-phosphate (M6P) signal in targeting newly synthesized lysosomal hydrolases to the lysosome, and how new lysosomes are formed. • Explain the difference between regulated and constitutive secretion. • Identify the structure and function of the ER, endosomes/phagosomes, glycocalyx, and clathrin coated vesicles. Be able to recognize them (if possible, for that organelle) on light and/or electron micrographs. • Examine the basic mechanisms of phagocytosis, pinocytosis, receptor-mediated endocytosis and autophagy. Be able to draw the pathway of the uptake of LDL/cholesterol via receptormediated endocytosis. • Define the basics of Botox, cystic fibrosis, and hypercholesterolemia Amir Mhawi 2 Endoplasmic Reticulum (ER) • The endoplasmic reticulum (ER) is organized into a mesh-like interconnected maze of branching tubes and flattened lamellae (sacs) rER sER • Two types: • Rough endoplasmic reticulum (rER) • Has ribosomes attached to its cytosolic surface • Smooth endoplasmic reticulum (sER) • Devoid (free) of attached ribosomes Amir Mhawi sER rER 3 Types of Ribosomes • Ribosomes are electron-dense particles made of two subunits of ribosomal rRNA and associated proteins • Special technique of transmission electron microscopy reveals many ribosomes attach to a single mRNA • Called polyribosome (or polysomes) • Polyribosomes are found in two locations in cytoplasm • 1) Free in the cytosol 2) Bound to rough ER TEM of free polyribosome Amir Mhawi 4 ER-bound and free polyribosomes lumen nucleus Electron-dense ribosomes attach to cytosolic surface of the rER, creating bound polyribosomes (in blue). Free polyribosomes are depicted in green. The single mRNA molecule, to which the ribosomes are attached cannot be revealed with this technique of TEM microscopy lumen lumen lumen Amir Mhawi 5 Rough Endoplasmic Reticulum (rER) • Consists of parallel stacks of membrane-limited flattened cisternae (plural of cisterna; sac) • The cytosolic surface of the limiting membrane is studded with ribosomes (hence the name rough ER) • Continuous with the outer membrane of the nuclear envelope (arrow in the TEM) • Well-developed in cells intensively engaged in protein synthesis • Plasma cells, fibroblast, pancreatic cells, etc..) • Presence of large number of attached ribosomes confer basophilia (affinity to basic dyes) to the cytoplasm of the protein-producing cells when viewed with the light microscope • Major functions: • Production proteins • Modification of the newly synthesized proteins • Quality control of the newly synthesized proteins basophilic cytoplasm 6 Light micrograph of a plasma cell Protein Synthesis by the rER-Bound Polyribosomes • Majority of proteins are synthesized by the polyribosomes bound to rough endoplasmic reticulum (rER) • Then proteins are packed into vesicles which move from rER to Golgi complex for further maturation • After maturation in Golgi, proteins are packed again into vesicles and transported to their destination: • Secretion outside the cell • Insertion in the plasma membrane or organelles’ membrane • Lumen of the ER and Golgi • Lysosomal matrix • This pathway called secretory pathway Amir Mhawi 7 • Proteins made on ER-bound polyribosomes need ER signal sequence • These Proteins are targeted to: • Outside the cell (secretion) • Plasma membrane • Organelles membrane • Lumens of the rER and Golgi • Lysosome matrix • Proteins made on free polyribosomes need organelle-specific targeting signal • These proteins are targeted to the cell organelles: • Nucleus (need NLS) • Mitochondrial matrix (need mitochondrial-targeting signal) • Peroxisome matrix (need peroxisomal-targeting signal) • Proteins of the cytosol need no targeting signal (e.g., enzymes, cytoskeletal subunits, etc..) 8 ER Signal Sequence and Signal Recognition Particle (SRP) Proteins made on ER-bound polyribosomes need ER signal sequence which direct the newly synthesized peptide to the lumen of the rER signal recognition particle (SRP): receptor for the ER signal sequence 1 2 3 4 6 5 Steps for entering the secretory pathway: 1- As the protein is translated, the ER signal sequence at amino terminus begins to protrude from the ribosome 2- SRP binds ER signal and this pauses translation 3- SRP carries the ribosome and the partially translated protein to rER membrane and binds the SRP receptor 4- This binding recruits the translocon channel, and then SRP disengages 5- Translation resumes, and the nascent protein is now threaded through the translocon into the ER lumen • Protein folds and is transported within a vesicle to Golgi 6- Ribosome subunits disassemble at the 3’-end of the mRNA Amir Mhawi 9 Insertion of Membrane Proteins (In Any Membrane of The Cell) • Membrane proteins, for anywhere in the cell (including mitochondria), are always translated on ER-bound polyribosomes 1 • Each has ER signal sequence at the amino terminus and membranespanning domain (20+ hydrophobic amino acids) (1) (Figure FYI) 2 3 4 • During the elongation of the polypeptide the ER signal sequence bound to the wall of the translocon channel while the rest of the of the polypeptide passes through the channel as a loop (2) • When the hydrophobic domain enters the translocon, the channel opens and releases the membrane protein into the rER membrane (3) • ER signal sequence is cleaved off by the signal peptidase (3) • The transmembrane protein is retained in the lipid bilaye (4)r 5 Membrane protein Top • The protein continues to grow at the cytosolic side until it reaches full length (4) • Then the membrane protein is transported by a vesicle to its destination (5; lower left illustration) • All plasma membrane ion channels, ligand receptors, etc. (as well as membrane proteins in other organelles) go through this secretory pathway Essential Cell Biology 1st Protein Modification in ER: Formation of Disulfide Bonds • Free sulfhydryl groups (SH) on cysteine residues are oxidized in ER lumen to form disulfide (S-S) bonds • Bonds form either: • Within same protein • Or link different proteins to form multisubunit complexes Disulfide bonds • Function: • Aids in folding, which stabilizes protein conformation • Multisubunit complexes of secreted (e.g., antibodies) or membrane proteins held together by disulphide bonds usually assemble in the rER Amir Mhawi Example of multisubunit complex assembled by disulfide bonds11 IgG antibody light and heavy chains 2nd Protein Modification in ER: Cleavage of ER Signal Sequence • N-terminal signal peptides are usually cleaved by signal peptidase (only in ER lumen) • After cleavage, the signal peptides are degraded peptidase Amir Mhawi 12 3rd Protein Modification in ER: N-Linked (initial) Glycosylation • Most nascent proteins in ER lumen receive Nlinked glycosylation at multiple sites on protein • N-linked polysaccharides attached to asparagine (asparagine amino acid symbol is N) • Many sugar molecules (multiple copies of mannose, glucose, N- acetylglucosamine) are pre-assembled, then transferred together in one step in rough ER to nascent protein Function of Glycosylation: • Aids protein folding (special chaperones in rER lumen bind attached sugars, use as “handles” to help fold proteins) • Slows rate of degradation of secreted proteins and membrane-bound proteins • Makes up recognition domains Amir Mhawi Image FYI 4th Protein Modification in ER: Protein Folding • Protein folding is aided by chaperones of the ER lumen such as BiP (binding protein) • BiP is a member of the heat shock proteins family • BiP is almost identical to cytosolic hsp70 (heat shock protein 70) • BiP binds the exposed hydrophobic patches in recently translated proteins to prevent their aggregation • After a protein has folded, its hydrophobic patches are buried within its structure • Folding ensures all the hydrophobic patches of amino acids are buried inside as the protein folds FUNCTION: • Correct folding and release from chaperones is necessary for exit of a protein from the rER Amir Mhawi 14 Exit from the ER is Controlled to Ensure Protein Quality • Correctly folded proteins exit ER in vesicles, transported to Golgi, and from there they are transported to their final destinations • Exit from ER is highly selective • Proteins fold up incorrectly in the ER are actively retained and not allowed to exit • Bind to resident chaperon proteins (figure A) • Misfolded proteins are dislocated back through the ER translocator channel and bound to ubiquitin protein to be degraded in the cytoplasm by proteasomes (figure B) Amir Mhawi A B removes N-linked carbohydrate 15 Clinical Correlation: Cystic Fibrosis (CF) An Example of Quality Control in rER • Quality control mechanism can be detrimental • Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (located on chromosome 7) results in slightly misfolded CFTR transmembrane protein • By far the most common mutation leading to cystic fibrosis is the deletion of phenylalanine at position 508 from CFTR • Normal CFTR protein is responsible for transporting chloride ion (Cl-) into and out of epithelial cells lining the respiratory tract • Mutated CFTR is retained in the ER, ubiquitinated (addition of ubiquitin protein), and marked for degradation by the proteosome in cytosol • Abnormal epithelial transport of Cl- affects the viscosity of the mucus covering the epithelial cells lining the respiratory tract • Cystic fibrosis is the most common fatal genetic disease in the Mhawi United States today, occurring in ~1 out of 3,300Amir live births 16 Clinical Correlation: Cystic Fibrosis (CF) cont’d • The defective Cl- channel protein in the bronchial epithelium causes decreased Cl- secretion and increased sodium ions Na+ and water reabsorption from the lumen • As a result, the “mucociliary escalator” malfunctions • With consequent accumulation of an unusually thick, viscous mucous secretion • The bronchioles become obstructed • Because fluids remain trapped in the lungs, individuals with cystic fibrosis have frequent respiratory tract infections Amir Mhawi 17 Smooth Endoplasmic Reticulum (sER) • The region of the endoplasmic reticulum lacking bound ribosomes • Arranged as interconnected channels • In the TEM section, sER appears as tubules and vesicles (described as tubule-vesicular; this image) • Well-developed in hepatocytes, steroid-producing cells, and striated muscle • Called sarcoplasmic reticulum in striated muscle Functions: • • • • Biosynthesis of steroid hormones Biosynthesis of membrane lipids Breakdown of toxins and drugs Sequestration of Ca++ in striated muscles nucleus L L L sER sER M M M sER L, lysosome; M, mitochondrion; sER, smooth endoplasmic reticulum TEM of two adjacent typical steroid-producing cell. The cytoplasm of thses cells is occupied by well-developed smooth endoplasmic reticulum (SER). The cells use cholesterol available in the lipid droplet (L) to make sterioid. Note the cristae of the 19 mitochondria (M). The cristae are in the form of tubules and vesiclesl. A, autophagosome; G, Golgi; N, nucleus; Golgi Apparatus • Located near the cell nucleus • Consists of a stacks of flattened membranebound sacs (cisternae) • Prominent in cells dedicated for protein production like fibroblasts and plasma cells • At the TEM level appears to consists of three defined regions: cis, medial, and trans Golgi • cis Golgi receives protein-loaded vesicles from ER • trans Golgi sorts matured proteins to their destinations • Proteins undergo further maturation during the transportation from cis to trans Golgi • At the LM level Golgi appears as a lightlystained area in the basophilic cytoplasm of the protein-producing cells Amir Mhawi Golgi apparatus Protein Modification in Golgi: Terminal Glycosylation 1. Modification of N-linked Sugars Image FYI • First, (high mannose) N-linked polysaccharides received in the ER are trimmed • Then, as the protein moves through the Golgi stacks new sugars are added back Cis Golgi Trans Golgi 2. O-linked Glycosylation • Polysaccharides are attached to the hydroxyl group of lipid and serine or threonine residues of proteins Amir Mhawi 21 Glycocalyx (AKA the Cell Coat) • The outer leaflet of the plasma membrane is covered with fuzzy layer of branched carbohydrates chains called glycocalyx • Carbohydrate chains attach to proteins form: • Glycoproteins • Carbohydrate chains attach to lipid form: glycocalyx • Glycolipids • Prominent in absorptive cells • e.g., cells lining the intestine, kidney tubules Function: • Cell recognition • cells of transplanted tissue/organ may be rejected due to the recognition of foreign integral membrane glycoproteins • Enzymes • Receptors Amir Mhawi 22 Trans-Golgi Network is the Sorting Station in the Secretory Pathway Cargo is sorted at the trans-Golgi network (TGN) into 3 different types of vesicles with different destinations: 1- Vesicles that bud off from trans Golgi network and become lysosomes (not shown in this image) 2- Regulated secretory vesicles • AKA regulated secretory pathway 3- Constitutive secretory vesicles • AKA constitutive secretory pathway Amir Mhawi 23 Vesicular Trafficking • Proteins and lipids synthesized in the ER are carried out (red arrows) by the transport vesicles to Golgi apparatus and from Golgi apparatus to other cellular compartments (e.g., late endosome then lysosome) or for secretion (a process called exocytosis) • Transport vesicles also bring extracellular molecules from the plasma membrane (green arrows), through the endosomes, to lysosomes for degradation (a process called endocytosis) • The formation of these vesicles is driven by the assembly of coat protein Amir Mhawi 24 Type of Vesicles 1- Clathrin-coated vesicle (see next slide): • Formed when vesicles are formed at trans-Golgi network and during receptor-mediated endocytosis • Clathrin is a triskelion (three-legged structure) that can be assembled into a polygonal lattice at the cytoplasmic face of the membrane • Clathrin-coated vesicle formation is initiated when a ligand (e.g., low density lipoprotein (LDL) molecules, which looks red in the next slide) is recognized by receptors located in the plasma membrane (yellow) • At this site, the plasma membrane (black line) is indented toward the cytoplasm and the indentation is coated with clathrin (blue) to form a clathrin-coated pit (stage 1) • Clathrin-coated pit progresses into a deep invagination (stage 2) that eventually gives rise to a clathrin-coated vesicle (stage 3), which is still connected to the plasma membrane by a neck • Dynamin (green scissors in stage 3) is an enzyme that pinches off and suspends the vesicle free in the cytoplasm(stage 4) Amir Mhawi to breakdown the LDL (stage is not shown) 25 • LDL-loaded free vesicle is directed to the lysosome Receptor-mediated endocytosis of LDL Amir Mhawi 26 dynamin CV: Clathrin-coated vesicle If: intermediate filament Clinical Correlation: Familial Hypercholesterolemia (FH) • A genetic disorder caused (almost always) by a defective LDL receptor (LDLR) or a defective apo-B protein • Apo B protein is a hydrophilic protein warping around LDL molecule so it can be carried through the blood and also helps with binding LDL image FYI molecule to its receptor • Characterized by very high levels of low-density lipoprotein (LDL) in serum • People who have one abnormal copy (are heterozygous) of the LDLR gene may develop cardiovascular disease prematurely at the age of 30 to 40 • Heterozygous FH is a common genetic disorder occurring in 1:500 people in most countries • Having two abnormal copies (being homozygous) may cause severe cardiovascular disease in childhood 28 Types of Vesicle cont’d 2- Coatomer-coated vesicles: • Vesicles are coated with protein that help with transportation between ER and Golgi • Two types of protein: • COP I: coating vesicles returning from Golgi to rER (retrograde transportation) • COP II: coating vesicle moving from rER to cis Golgi (anterograde transportation) capillary lumen 3- Caveolin-coated vesicles (red arrows in TEM): • Found in endothelial and smooth muscle cells surrounding connective tissue 29 Machinery Required to Fuse Vesicular membrane with the Target membrane: SNARES SNARE stands for SNAP Receptor • V-SNARES present on Vesicles • T-SNARES present on Target membranes • There are at least 35 different SNARES in the cell, each associated with a particular membraneenclosed organelle ex. rER, cis-Golgi, medial Golgi, trans-Golgi, plasma membranes, early endosomes, late endosomes, lysosomes etc. Function: • Target vesicles to correct membrane and docks them • Are the primary proteins that drive the fusion reaction Amir Mhawi 30 Clinical Correlation: BOTOX • Botox injections are the best known of a group of medications that use various forms of botulinum toxin to temporarily paralyze muscle activity • Purpose is to prevent wrinkles when injected into the musculature of the forehead • Botox degrades specific SNARES • This degradation prevents fusion of exocytic vesicles containing muscle-stimulating neurotransmitter (acetylcholine) with the plasma membrane • Prevents muscle contraction Amir Mhawi FYI Machinery for Vesicle Targeting: RABS (Small GTP-Binding Proteins) • RABs Contribute to specificity of docking reaction • ~70 different Rabs have been identified in humans, each localized to a specific membrane • A Rab on the vesicle binds a tethering protein on the target membrane • Gets the two membranes close, and then SNARES bind and fuse the two membranes Amir Mhawi 32 Lysosomes • Lysosomes are the digestive compartment of the cell • Contain a large variety of acid hydrolytic enzymes nucleus • Become active only in acidic pH • Found in all cell types • Profound in phagocytic cells lysosomes • Macrophages • Neutrophils Amir Mhawi 33 Biogenesis of Lysosomes • Synthesis of lysosomal enzymes (hydrolases) occurs in the rER • All lysosomal hydrolases receive a special signal as they travel through the rER and Golgi • Mannose-6-phosphate (M6P) signal • Membranes of trans Golgi network contain M6P receptors • Receptors bind lysosomal hydrolases and cluster them into clathrin-coated vesicles • Clathrin-coated vesicles bud off trans Golgi network and transported to earl/late endosome • Late endosome matures to lysosome when its pH becomes acidic • Enzymes deficient of M6P fail to reach the lysosomes • This will result in the accumulation of undigestible molecules in the lysosomes • AKA LYSOSOMAL STORAGE DISEASES • Inclusion-Cell disease (I-cell disease) • Glycogen storage disease • Tay-Sachs disease, etc… Figure detail FYI 34 Source of Substrates for Lysosomal Degradation Material destined for degradation in the lysosome (mostly) comes from 3 sources: 1 1. Endosomes (from endocytosis) 2. Phagosomes (from phagocytosis) (the above 2 will be covered at the end of the lecture) 2 3 3. Autophagosomes (from autophagy) Amir Mhawi Figure details FYI Source of Substrates for Lysosomal Degradation (cont’d) Autophagosomes (from autophagy) • Mechanism by which the cell degrades its own organelles or part of the cytoplasm if they become old or non-functional (e.g., mitochondria or sER) • When a patient stops taking phenobarbital, the excess sER is destroyed by autophagy • The cell can degrade its own organelle during starvation to provide necessary nutrient • Material to be degraded is wrapped in membrane (source of membrane is controversial) • The formed structure called autophagosome • Autophagosome is directed toward the lysosome Autophagosome containing peroxisome and mitochondrion Amir Mhawi 36 ENDOCYTOSIS • The cell has multiple ways of bringing in substances from the extracellular fluid: 1- Phagocytosis (“cell eating”)- ingestion of large particles such as microorganisms and cell debris via large vesicles called phagosomes 2- Pinocytosis (“cell drinking”)- ingestion of fluid and molecules via small vesicles 3- Receptor-mediated endocytosis (covered earlier) • All are forms of endocytosis Amir Mhawi “cell eating” “cell drinking” 37 PHAGOCYTOSIS-”CELL EATING” • Solid particles like bacteria are normally coated with antibodies and marked for destruction (“eat me” signal) • This process called opsonization (figures a and c) • Specialized cells (phagocytes, neutrophils) extend pseudopodia (via actin microfilament polymerization) toward the opsonized particle and engulf it inside a phagosome • Phagosome then fuses with a lysosome for digestion of the particle • Note that there is no need to opsonize nonbiological material (e.g., carbon particles or tattoo pigments (figure b) • Carbon and tattoo pigments cannot be digested inside the lysosomes and remain as residual body Amir Mhawi c Phagocytosis of a bacteria by a neutrophil PINOCYTOSIS- “CELL DRINKING” • An old term referring to the fluid intake by the cell • Can be tested by adding fluorescent dye to the extracellular fluid • Fluorescent dye within a drop of extracellular fluid will be internalized by a vesicle • Two examples of the pinocytosis: 1) caveolae 2) clathrin-coated vesicles Amir Mhawi 39 Exocytosis • The process by which the contents of a cargoloaded vesicle are released to the exterior through fusion of the vesicular membrane with the cell membrane is called EXOCYTOSIS. • Two secretory pathways: • Regulated and constitutive secretory pathways Regulated secretory pathway • The cargo-loaded vesicles will be stored in cytoplasm, not exocytosed until proper time • Content is released only when the cell receive a proper signal: • e.g., insulin, pancreatic enzymes, neurotransmitters • Rise in cytosolic Ca++ is the signal for the movement and fusion of the vesicle with the plasm membrane Amir Mhawi EM of pancreatic acinar cell showing regulated secretory vesicles (arrowhead). These vesicles contain digestive enzymes that 40 are only released after eating food Constitutive secretory pathway • Cargo-loaded vesicle of the constitutive secretory pathway travel to the plasma membrane and fuse with it immediately after leaving trans Golgi • No need to store in the cytoplasm • Vesicles require No signal to fuse with the plasma membrane or to release the content • Example: • Albumin produced by the liver • Collagen produced by fibroblasts • Antibodies produced by plasma cells Amir Mhawi 41 END OF THE LECTURE! Extra reading if so desired: Relevant portion of “Cytoplasmic Organelles” of Chapter 2 “The Cytoplasm” Junqueira’s Basic Histology Amir Mhawi 42