BIOM-2131 Introductory Molecular Biology Intracellular Compartments And Protein Transport (part 2) LECTURE OVERVIEW ▪ 16.1 Vesicular Transport ▪ 16.2 Secretory pathway ▪ 16.3 Endocytic pathway LECTURE 16 Growth and maintenance of organelles… • … requires not only import of new proteins, but also incorporation of new lipids into the organelle membranes • for some organelles, phospholipids are imported by lipid-carrying proteins at junctional complexes • organelles that are part of endomembrane system can receive lipids via transport vesicles Alberts, et. al, Essential Cell Biology, 6th edition, Figure 15-17 (partial) p. 531 Vesicular transport • extends outward from the ER to PM • reaches inward from the PM to ER • routes of communication between the interior of the cell and its surroundings Alberts, et. al, Essential Cell Biology, 6th edition, Figure 15-18, p. 532 Vesicular transport • ER is usually the 1st step on the pathway to another destination • next stop - Golgi • proteins and lipids are modified and sorted • continual budding and fusion of transport vesicles Alberts, et. al, Essential Cell Biology, 6th edition, Figure 15-19 (partial), p. 532 Vesicular transport • between membrane-enclosed compartments of the endomembrane system is highly organized • endocytic pathway • secretory pathway Alberts, et. al, Essential Cell Biology, 6th edition, Figure 15-19 (partial), p. 532 Vesicular transport • coated vesicles • cells produce several kinds of coated vesicles, each with a distinct protein coat on their cytosolic surface • after budding from its parent organelle, the vesicles shed its coat allowing the membrane to interact directly with the membrane to which it will fuse Clathrin-coated vesicles … • …. bud from PM and Golgi • start as clathrin-coated pit • clathrin molecules assemble into a basketlike network • this assembly process starts shaping membrane into a vesicle Alberts, et. al, Essential Cell Biology, 6th edition, Figure 15-21 (partial), p. 534 Clathrin-coated vesicles transport selected cargo • help to select the cargo molecules for transport • different types • transport signal • cargo receptor Alberts, et. al, Essential Cell Biology, 6th edition, Figure 15-21 (partial), p. 534 • GTP-binding protein Some types of coated vesicles • other kinds of transport vesicles, with different coat proteins, are also involved in vesicular transport Alberts, et. al, Essential Cell Biology, 6th edition Table 15-4, p. 535 Vesicle docking • transport vesicles are actively transported by motor proteins that move along cytoskeletal fibers • once the destination is reached, the vesicle must recognize and dock with its specific organelle prior to fusion of membranes and unloading of the cargo • molecular markers identifying origin and cargo of a vesicle Proteins involved in vesicle docking • Rab proteins • diverse family of monomeric GTPases • specific; surface of each type of vesicle • recognized by corresponding tethering proteins on the cytosolic surface of the target membrane • Additional recognition: • TM proteins called SNAREs • v-SNAREs • SNAREs on the vesicle • t-SNAREs • SNAREs on the target membrane • firmly dock the vesicle in place Directing transport vesicle to their target membrane • final fusion of the two membranes • catalyzed by complementary SNAREs Alberts, et. al, Essential Cell Biology, 6th edition Figure 15-22, p. 535 SNARE proteins catalyze the fusion of the vesicle and target membranes • all membrane fusions must be catalyzed by specialized proteins that assemble to form a fusion complex that provides means to cross energy barriers • two bilayers must come within 1.5 nm • lipids can intermix • delivery of cargo + addition of the vesicle membrane to the membrane of the organelle SNARE proteins catalyze the fusion of the vesicle and target membranes • after fusion, the SNAREs are pried apart to be reused again Alberts, et. al, Essential Cell Biology, 6th edition Figure 15-23, p. 536 Secretory Pathways • each molecule that travels along this secretory pathway • passes through a fixed sequence of membrane-enclosed compartments • often chemically modified en route Alberts et al, Essential Cell Biology; Figure 15-19 (partial); p. 532 Most proteins are covalently modified in the ER • disulfide bond formation • rxn catalyzed by enzyme resident to ER lumen • formed by oxidation of pairs of Cys side chains • help to stabilize the structure of the proteins • why do disulfide bonds not form in cytosol? • reducing environment Most proteins are covalently modified in the ER • glycosylation • rxn catalyzed by glycosylating enzymes present in the ER • conversion to glycoproteins by covalent attachment of short, branched oligosaccharide side chains composed of multiple sugars • help to stabilize the structure of the proteins • are proteins in cytosol glycosylated? • very few • only a single sugar attached Oligosaccharides on the proteins • serve various functions • can protect a protein from degradation • hold the protein in ER until is properly folded • help guide the protein to the appropriate organelle by serving as a transport signal for packaging into appropriate transport vesicles • displayed on the cell surface • cell’s outer carbohydrate layer • glycocalyx How are oligosaccharide side chains added in the ER? 1/2 • individual sugars are not added one by one to the protein • branched oligosaccharide containing a total of 14 sugars is attached en bloc • the oligosaccharide is originally attached to a specialized lipid, dolichol • then transferred to the amino (NH2) group of asparagine N-glycosylation • immediately after a target Asn emerges in the ER lumen during protein translocation How are oligosaccharide side chains added in the ER? 2/2 • addition takes place in a single enzymatic step • oligosaccharyl transferase • N-linked glycosylation • most common type of linkage found on glycoproteins • glycosylated asparagine • Asn-X-Ser • Asn-X-Thr Alberts, et. al, Essential Cell Biology, 6th edition, Figure 15-24, p. 537 Once the sugar moiety is added to the protein • the addition of the 14-sugar oligosaccharide in the ER is only the first step in series of further modifications • attached oligosaccharides undergo extensive modifications oligosaccharide processing • begin in the ER and • continues in the Golgi • N-linked oligosaccharides on mature glycopeptides are remarkably diverse What happens to proteins made in ER? • some proteins are destined to function in ER • C-terminal sequence of 4 aa called ER retention signal • this ER retention signal is recognized by membrane-bound receptor protein in the ER and Golgi • what happens if these proteins leave ER? • returned to ER should they manage to escape into Golgi Quality control prior to exit from ER • most proteins are destined for other locations • will be packaged into transport vesicles • proteins actively retained in the ER by binding to chaperone proteins • proteins that fail to fold correctly • dimeric/multimeric proteins that do not assembled correctly Quality control prior to exit from ER • chaperones …. Alberts, et. al, Essential Cell Biology, 6th edition, Figures 4-8 (partial), 4-9 (partial), p. 127-128 Chaperones… • prevent misfolded proteins from aggregating • help steer proteins along path toward proper folding • misfolded proteins • exported to the cytosol • degraded by the proteosome Exit from the ER is controlled to ensure protein quality • Ab are assembled into complete Ab molecule in the ER • partially assembled Ab are retained in the ER until all four polypeptide chains are assembled • any Ab molecule that fails to assemble properly is degraded The size of ER is controlled by the demand for protein folding • chaperone quality control system can become overwhelmed • misfolded proteins accumulate in the ER • trigger complex program called • unfolded protein response • slow synthesis of additional proteins • expand ER and boost production of proteins involved in quality control and proper protein folding Accumulation of misfolded proteins in the ER lumen triggers…. • … unfolded protein response • TM sensor proteins • activation of chaperone genes • enhance proteinprocessing capacity of the ER • promote protein degradation • inhibit protein synthesis Alberts et al, Essential Cell Biology; Figure 15-25 (partial); p. 539 Unfolded protein response allows a cell to …. • … adjust the size of its ER to properly handle the volume of proteins entering the secretory pathway • cell directed to self-destruct by undergoing apoptosis if expanded ER cannot keep up • such situation may occur in adult-onset diabetes • tissues gradually become resistant to the effect of insulin more insulin secreted The Golgi Apparatus… • consists of a collection of flattened, membraneenclosed sacs called cisternae • each stack contains 3-20 cisternae • # of Golgi stack per cell varies greatly • depends on cell type • each Golgi stack has two distinct faces • an entry, or cis, face • adjacent to ER • an exit, or trans, face • points toward the PM Golgi apparatus • the outermost cisterna at each face is connected to a network of interconnected membrane tubes and vesicles Alberts et al, Essential Cell Biology; Figure 15-26 (partial); p. 539 Golgi Apparatus • enter the cis Golgi network via transport vesicles derived from the ER • proteins then travel through the cisternae in sequence in two ways: Golgi Apparatus • by means of transport vesicles • bud from one cisterna and fuse with the next • by maturation process • the Golgi cisternae themselves migrate through the Golgi stack • proteins exit from the trans Golgi network in transport vesicles • destined to PM • other organelle of endomembrane system Golgi - Protein sorting • both, the cis and trans Golgi networks, are important for protein sorting • cis Golgi network: • either allow proteins to move onward through the Golgi stack • or if proteins contain ER retention signal, return to ER • trans Golgi network: • sorted by destination • PM, lysosomes (via endosomes) Golgi – Processing of oligosaccharide chains • many of the oligosaccharide chains added to the proteins in ER undergo further modifications in the Golgi • on some proteins, more complex oligosaccharide chains are created by highly ordered process as the protein passes through the Golgi stacks Secretory proteins are released from the cell by exocytosis Alberts et al, Essential Cell Biology; Figure 15-19 (partial); p. 532 Secretory proteins are released from the cell by exocytosis • constitutive exocytosis pathway • supplies the PM with newly made lipids and proteins • carries soluble proteins to the cell surface to be released to the outside • process called secretion • entry does not require a particular signal sequence • operates continually in all eukaryotic cells Secretory proteins are released from the cell by exocytosis • regulated exocytosis pathway • operates only in cells that are specialized for secretion • proteins have special surface properties that cause them to aggregate with one another under conditions that prevail in trans Golgi network • acidic pH, high [Ca2+] • selective aggregation allows secretory proteins to be packaged at up to 200-fold increase in concentration Secretory proteins are released from the cell by exocytosis • regulated exocytosis pathway • each specialized secretory cell produces large quantities of a particular product • stored in secretory vesicles for later release • bud off from trans Golgi network and accumulate near PM • wait for extracellular signal Constitutive and regulated secretory pathways Alberts et al, Essential Cell Biology; Figure 15-30; p. 543 What happens at the PM? • when a secretory vesicle or transport vesicle fuses with PM… • discharges its content by exocytosis • its membrane becomes a part of the PM • do these incorporations greatly increase the surface area of the PM? • transient • membrane components are removed by endocytosis • membrane retrieval pathways Tracking protein and vesicle transport… in a test tube… 1/2 • how proteins shuffle from one cell compartment to another • in a test tube • protein bearing a signal sequence + preparation of isolated organelles • test to see if protein is taken up by the organelle • produce protein in vitro by cell-free translation of a purified mRNA encoding the polypeptide • use radioactive aa to label protein Tracking protein and vesicle transport… in a test tube… 2/2 + • centrifugation • add protease • add detergent Alberts et al, Essential Cell Biology; Panel 15-27; p. 541 Tracking protein and vesicle transport… in mutant yeast… • temp-sensitive mutant yeast cells • at 25C function normally • at 35C proteins inactivated • defective for secretion at high temp Alberts et al, Essential Cell Biology; Figure 15-28; p. 542 Lab 4: GFP-tagged protein • during your lab, you used genetic engineering to design your protein tagged with a green fluorescent protein • most common method for tracking a protein as it moves through the cell • fortunately, for many proteins studied, the addition of GFP to the N- or C-terminus does not perturb the protein’s normal structure, function or transport http://www.synthesisgene.com/vector/pEGFP-N3.pdf Alberts et al, Essential Cell Biology; Figure 15-29; p. 542 Endocytic pathways • eukaryotic cells are continually taking up fluid along with large and small molecules by process of endocytosis Alberts et al, Essential Cell Biology; Figure 15-19 (partial); p. 532 Endocytic pathways • certain specialized cells are able to internalize large particles and even other cells • the material to be ingested is progressively enclosed in a small portion on PM endocytic vesicle • ingested material + membrane components delivered to endosomes • recycled to PM or send to lysosomes Main types of endocytosis • pinocytosis • involves ingestion of fluid and molecules via small pinocytic vesicles (<150 nm in ) • phagocytosis • involves ingestion of large particles, such as microorganisms and cell debris, via large vesicles called phagosomes (<250 nm in ) Specialized phagocytic cells ingest large particles • in protozoa (unicellular eukaryotes) • ingest large particles, such as bacteria, by taking them up into phagosomes • phagosomes then fuse with lysosomes • few cells in multicellular organism • phagocytic cells • macrophages, neutrophils • defend against infection by ingesting invading microorganisms Phagocytosis by…. • to be taken up by macrophages or neutrophils • particles must first bind to the phagocytic cell surface and activate one of a variety of surface receptors • some of these receptors recognize Ab • binding induces the phagocytic cell to rearrange its cytoskeleton to extend sheetlike projections of the PM, called pseudopods, that engulf the bacterium • pseudopods fuse = phagosome Alberts et al, Essential Cell Biology; Figure 15-32 (partial); p. 545 Phagocytosis by…. • phagocytic cells also play important role in scavenging dead and damaged cells and cell debris • macrophages ingest each day more than one billion worn-out RBC in the human body Alberts et al, Essential Cell Biology; Figure 15-32 (partial); p. 545 During the process of pinocytosis… • … eukaryotic cells continually ingest bits of their PM along with extracellular fluid • rate of PM internalization varies among cell types • e.g. macrophage • swallows 25% of its own volume of fluid each hour • removes 3% of its PM each minute, or 100% in about ½ hour • e.g. fibroblast • occurs more slowly • cell’s total surface area and volume remain unchanged Pinocytosis is carried out mainly by… • … the clathrin-coated pits and vesicles • after they pinch off PM, they are internalized and delivered to endosomes • indiscriminate • simply trap any molecule that happen to be present in the extracellular fluid and carry them inside the cell Pinocytosis …. • … can sometimes be more selective • presence of complementary receptors • provide selective concentrating mechanism that increases the efficiency • …. receptor-mediated endocytosis • receptor-macromolecule complexes in clathrin-coated vesicles Receptor-mediated endocytosis • cholesterol is transported bound to protein, forming particles, such as low-density lipoproteins, LDLs Alberts et al, Essential Cell Biology; Figure 15-33; p. 546 Receptor-mediated endocytosis • take up many other essential metabolites, such is vitamin B12 and iron • are required to make hemoglobin • enter immature RBCs as part of a complex with their respective receptor proteins • exploited by viruses • influenza virus, HIV • gain entry into cells Alberts et al, Essential Cell Biology; Figure 15-34 (partial); p. 547 Endocytosed macromolecules are sorted in endosomes • endosomal compartments • complex set of connected membrane tubes and larger vesicles • early endosomes • just beneath the PM • late endosomes • located closer to the nucleus • early endosomes mature gradually into late endosomes as they fuse with each other or with a preexisting late endosome • lysosome Endosomes • interior of the endosome is • acidic (pH 5-6) • ATP-driven H+ pump in the endosomal membrane • pumps protons into the endosome lumen from the cytosol • major sorting station in the inward endocytic pathway • due to acidic environment, many (but not all) receptors release their bound cargo Endosomes • receptors inside of the endosome • their fate differ on the type of the receptor • most are returned to PM – same domain • some travel to lysosomes • some proceed to a different domain of the PM • transfer their bound cargo molecules across the cell from one extracellular space to another • transcytosis Fate of receptor proteins following their endocytosis Alberts et al, Essential Cell Biology; Figure 15-35; p. 547 Lysosomes • principal site of intracellular digestion • membrane delineated • contain ~ 40 types of hydrolytic enzymes • controlled intracellular digestion of: • extracellular material • worn-out organelles Alberts et al, Essential Cell Biology; Figure 15-36 (modified); p. 548 Lysosomes • unique membrane • ATP-driven H+ pump • metabolite transporters: • aa, sugars, nt • most of the lysosomal membrane proteins are unusually highly glycosylated • sugars which cover most of the protein surfaces facing the lysosome lumen protect the proteins for digestion by the lysosomal proteases Alberts et al, Essential Cell Biology; Figure 15-36 (modified); p. 548 Tagging and sorting of lysosomal enzymes • specialized digestive enzymes and membrane proteins of the lysosome • synthesized in ER → transporter through Golgi to the trans Golgi network → recognized by specific receptor → packed into transport vesicles • in cis Golgi network tagged with mannose-6-phosphate • receptor for mannose-6-phosphate Materials destined for degradation in lysosomes… • … follow different pathways to the lysosome Alberts et al, Essential Cell Biology; Figure 15-37; p. 549 Materials destined for degradation… • autophagy • used to degrade obsolete parts of the cell • process involves enclosure of the organelle by double membrane autophagosome → fuses with a lysosome • cannibalistic form of digestion Lecture 16 Reading • Chapter 15: Intracellular compartment and protein transport • pages 532 - 552 Upcoming: Lecture 17 Reading • Chapter 16: Cell Signaling • pages 553 - 565
