Ch.15, Part 2 - Cell Compartments Flashcards
- Vesicular Transport - Secretory Pathways - Endocytic Pathways
Provide a general overview of the major outward secretory pathway.
Major outward secretory pathway - starts w synth of proteins on ER mem → entry into ER → thru Golgi → cell surface OR side branch leading thru endosomes to lysosomes.
Provide a general overview of the major inward endocytic pathway.
Major inward endocytic pathway - responsible for ingestion and degradation of EC molecules; moves materials fr pmem → thru endosomes → lysosomes.
Ea xprt vesicle must take w it only the proteins approp to its destination and must fuse only w approp target mem, thereby maintaining the distinct protein/lipid composition of organelles. What feature of xprt vesicles enables this?
Proteins displayed on surface of xprt vesicles ensures proper vesicle cargo/destination → maintains distinct protein/lipid comp of organelle.
Vesicle budding is driven by the assembly of a ________.
Vesicle budding is driven by the assembly of a protein coat.
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Coated vesicles - vesicles w distinctive protein coat on cytosolic surface; bud fr mems.
- Bud fr parent organelle → vesicle sheds coat → allows vesicle mem to interact directly w target mem.
- Several types of coated vesicles, ea w distinctive protein coat.
- Coat helps shape mem into a bud and captures molecules for onward xprt.
Coated vesicles are those w distinctive protein coat on their _______ (cytosolic/non) surface → bud fr parent organelle → vesicle sheds coat → allows vesicle to ______________________.
Coated vesicles are those w distinctive protein coat on their cytosolic surface → bud fr parent organelle → vesicle sheds coat → allows vesicle to interact directly w target mem.
- Coat helps shape mem into a bud and captures molecules for onward xprt.
________-coated vesicles are v well studied and bud fr both Golgi on outward secretory pathway and fr pmem on inward endocytic pathway.
Clathrin-coated vesicles - well-studied; bud fr both Golgi on outward secretory pathway and fr pmem on inward endocytic pathway.
Describe the endocytic mechanism via clathrin-coated vesicles.
Endocytic mechanism:
- At pmem, ea vesicle starts off as a clathrin-coated pit → clathrin molecules assemble into a basket-like network on cytosolic surface of mem → assembly process starts shaping mem into a vesicle.
- A small GTP-binding protein (dynamin) assembles as a ring around neck of ea deeply invaginated coated pit → dynamin and assembly proteins causes ring to constrict → pinch off vesicle fr parent mem.
- Other xprt vescles (w diff protein coats) form in similar way.
________ are a second class of coat proteins that secure the clathrin coat to the xprt vesicle mem and help select cargo molecules for xprt.
Adaptins are a second class of coat proteins that secure the clathrin coat to the xprt vesicle mem and help select cargo molecules for xprt.
- Diff types of adaptins for binding diff types of cargo receptors in diff organelles.
T/F: The protein coat itself plays no part in choosing specific molecules for xprt.
True
The protein coat itself plays no part in choosing specific molecules for xprt.
- Adaptins - a second class of coat proteins; secure clathrin coat to vesicle mem and help select cargo molecules for xprt.
Molecules for onward xprt (beyond ER) carry specific xprt signals that are recog by __________ in Golgi/pmem → _______ help capture specific cargo molecules by trapping the _________ that bind them.
Molecules for onward xprt (beyond ER) carry specific xprt signals that are recog by cargo receptors in Golgi/pmem → adaptins help capture specific cargo molecules by trapping the cargo receptors that bind them.
- Thus, a selected set of cargo molecules, bound to their specific receptors, is incorporated into the lumen of ea newly formed clathrin-coated vesicle.
_____-coated vesicles are another class of coated vesicles; involved in xprt b/w ER and Golgi, and fr one part of Golgi to another.
COP-coated vesicles (COP = “coat protein”) - another class of coated vesicles; involved in xprt b/w ER and Golgi, and fr one part of Golgi to another.
After budding fr a mem, vesicles are often actively xprtd by ________ that move along cytoskeletal fibers
After budding fr a mem, vesicles are often actively xprtd by motor proteins that move along cytoskeletal fibers
Vescile docking deps on ______ and ______.
Vescile docking deps on tethers and SNAREs.
Vesicles identify, bind, and fuse w target organelle via specific surface markers on both the vesicle surface and target mem, incl pmem. What types of proteins are involved in this process?
- Rab proteins - diverse family of monomeric GTPases; specific to ea type of vesicle.
- Tethering proteins - filamentous protein on cytosolic surface of target mem; recog/bind specific Rab proteins.
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SNAREs - family of xmem proteins; involved in vesicle recog, ensure proper docking, help catalyze fusion and deliver soluble contents into organelle, and add vesicle mem to target mem.
- v-SNAREs = SNAREs on vesicles
- t-SNAREs = SNAREs on target mem
Rab proteins, tethering proteins, and SNAREs help direct xprt vesicles to their target mems. Describe the tethering, docking, and fusion processes.
- Tethering - tethering protein on target mem recogs/binds vesicles bearing approp Rab protein → tethering protein guides vesicle toward target mem.
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Docking - v-SNAREs binds complementary t-SNAREs → firmly docks vesicle in place.
- Docking reqs only that the two mems come close enough for SNAREs to interact.
- Fusion - v- and t-SNAREs catalyze final fusion of vesicle and target mems.
- Post-fusion - SNAREs unwind → reused.
Rab proteins, tethering proteins, and SNAREs help direct xprt vesicles to their target mems. After tethering and docking, describe why the two mems don’t spontaneously fuse t/g.
Fusion - v-/t-SNAREs catalyze final fusion of vesicle and target mems. Sometimes reqs special stim signal:
- Fusion reqs much closer approach: bilayers must come w/i 1.5 nm of ea/o so that their lipids intermix → water must be displaced fr hphilic surfaces of mems (highly energ unfav) → prevents mems fr fusing randomly.
- Thus, all mem fusions must be catalyzed by specialized proteins (SNAREs): SNAREs form fusion complex (wrap around ea/o) → winding acts like a winch, pulling bilayers in close proximity and squeezing out any remaining water → enables random/spont fusion of mems.
The budding of clathrin-coated vesicles fr euk pmem fragments can be observed when adaptins, clathrin, and dynamin-GTP are added to the mem preparation. What would you observe if you omitted adaptins?
Clathrin coats cannot assemble in absence of adaptins that link clathrin to mem. At high clathrin concens and under approp ionic conditions, clathrin cages assemble in solution, but they are empty shells, lacking other proteins, and they contain no mem.
- This shows that the info to form clathrin baskets is contained in teh clathrin molecules themselves → can self-assemble.
The budding of clathrin-coated vesicles fr euk pmem fragments can be observed when adaptins, clathrin, and dynamin-GTP are added to the mem preparation. What would you observe if you omitted clathrin or dynamin?
- W/o clathrin - adaptins still bind to receptors in mem, but no clathrin coat can form and thus no clathrin-coated pits or vesicles are produced.
- W/o dyanmin - Deeply invaginated clathrin-coated pits form on the mem, but they do not pinch off to form closed vesicles.
The budding of clathrin-coated vesicles fr euk pmem fragments can be observed when adaptins, clathrin, and dynamin-GTP are added to the mem preparation. What would you observe if the pmem fragments were fr a prok cell instead?
Prok cells do not perform endocytosis → does not contain any receptors w approp cytosolic tails that could mediate adaptin binding → no clathrin can bind and no clathrin coats can assemble.
Most proteins are covalently modified in the ________.
Most proteins are covalently modified in the ER.
T/F: most proteins that enter ER are chemically modified there.
True
Most proteins that enter ER are chemically modified there.
- E.g. disulfide bonds, glycosylation.
Most proteins that enter ER are chemically modified there. One such mod is disulfide bonds. Describe how these bonds form and what purpose they serve.
Disulfide bonds - enzyme in ER lumen catalyzes oxidation of pairs of cysteine side chains → help stabilize struc of proteins that will encounter degradative (oxidative) enzymes and changes in pH outside cell (e.g. both excreted proteins and those in EC side of pmem).
- Recall: disulfide bonds do not form in cytosol bc more reducing agents and stable pH.
Glycosylating enzymes in the ________ catalyze the _________ (covalent/non) attachment of short, branched _________ side chains to proteins in ER lumen/mem → converted to _________.
Glycosylating enzymes in the ER catalyze the covalent attachment of short, branched oligosaccharide side chains to proteins in ER lumen/mem → converted to glycoproteins.
Glycosylating enzymes in the ER catalyze the covalent attachment of short, branched oligosaccharide side chains to proteins in ER lumen/mem → converted to glycoproteins. What functions do glycoproteins serve?
Various functions:
- protect protein fr degradation
- hold protein ER until properly folded
- help guide protein to target organelle by serving as xprt signal for packaging protein into approp xprt vesicles
- for EC-side mem proteins, oligos contrib to glycocalyx (carb layer) and help cell-cell recog.
T/F: Most cytosolic proteins are glycosylated.
False
V few cytosolic proteins are glycosylated; if they are, only single sugar attached.
- Recall: glycosylating enzymes in ER—not cytosol—catalyze covalent attachment of short branched oligo side chains to proteins in ER lumen/mem → converted to glycoproteins.
Where are the oligosaccharides located prior to attachment during glycosylation?
Oligos are originally attached to specialized lipid (dolichol) in ER mem.
T/F: During glycosylation, oligos are attached one-by-one to the approp glycosylation site.
False
Oligos are attached en bloc (as preformed branch of 14 sugars) to proteins w approp glycosylation site immediately as it emerges into ER lumen during protein xlctn.
The glycosylation site is a simple seq of ___ (#) AAs, one of wh is ________.
The glycosylation site is a simple seq of three AAs, one of wh is asparagine (asn, N).
- I.e. all asn’s are not glycosylated, must be part of partic tripeptide seq: either asn-X-Ser or asn-X-thr; X is almost any AA; Serine = ser, S; Threonine = thr, T.
Glycosylation is catalyzed by a _______ (cytosolic/mem-bound/lumenal) enzyme in ______ (mult/one) enzymatic step.
Glycosylation is catalyzed by mem-bound enzyme in a single enzymatic step.
- Oligosaccharyl transferase has active site exposed on lumenal side of ER mem, wh explains why cytosolic proteins are not glycosylated in this way.
- N-linked oligo side chains - those linked to asparagine (asn, N) NH2 group; by far most common type.
__-linked oligo side chains are by far the most common type of glycoprotein.
N-linked oligo side chains are by far the most common type of glycoprotein.
- N-linked oligo side chains - those linked to asparagine (asn, N) NH2 group.
T/F: N-linked oligos on mature glycoproteins are identical.
False
N-linked oligos on mature glycoproteins are v diverse.
- Glycosylation is merely first step in series of further mods before mature glycoprotein reaches cell surface.
Summarize the glycosylation mechanism.
Glycosylation mechanism:
- Polypeptide chain grows thru translocator channel as large loop; oligosaccharide (~14 branched sugars) is attached to dolichol—a specialized lipid in ER mem (adj to xlctr channel)…
- Glycosylation site (tripeptide seq w asparagine) enters ER lumen.
- Oligosaccharyl transferase catalyzes xfr of oligo fr dilochol to asparagine (asn, N) side chain.
- Almost always linked to asn’s NH2 group → “N-linked”.
Exit fr the ER is controlled to ensure protein quality. Describe how chaperone proteins are involved in this QC process.
Chaperone proteins in ER bind misfolded proteins and improperly assembled di/multimeric proteins → retain until proper folding/assembly occurs.
- Prevents misfolded proteins from aggregating → helps steer toward proper folding.
- If proper folding/assembly still fails → exported to cytosol → degraded.
ER quality control can be overwhelmed by an over-accumulation of misfolded proteins in the ER. What mechanism is triggered in response to this accumulation?
ER quality control can be overwhelmed → misfolded proteins accumulate in ER → if accumulation is large enough, triggers unfolded protein response (UPR) → initiates prod of more ER, incl more chaperones and other proteins involved w quality control.
- UPR allows cell to adjust ER size dep on load of proteins entering secretory pathway.
- If ER size ↑ fails to cope w quality control load → UPR initiates apoptosis.
The unfolded protein response (UPR) allows the cell to adjust ER size dep on load of proteins entering secretory pathway. What happens if increasing the size of ER (and chaperones/other proteins) still doesn’t sufficiently cope w the protein load?
If ER size ↑ fails to cope w quality control load → UPR initiates apoptosis.
- E.g. adult-onset diabetes - tissues gradually become resistant to effects of insulin → insulin-secreting cells in pancreas produce more and more insulin → ER eventually reaches max capacity → UPR can trigger cell death → # insulin-secreting cells ↓ → load on remaining cells ↑ → feed-forward and disease exacerbates.
Describe the UPR mechanism.
Misfolded proteins are recognized by several types of xmem sensor proteins in the ER mem, ea of wh activates diff part of UPR. Describe two such scenarios.
UPR mechanism:
- some misfolded proteins stim prod of xcr regulators → activate genes encoding chaperones/other ER QC proteins.
- other misfolded proteins inhibit protein synth → ↓ flow of proteins thru ER.
Proteins are further modified and sorted in the Golgi. Describe its structure and typ location.
Golgi - stacks of flattened, mem-enclosed sacs (cisternae); ea stack contains 3–20 cisternae, dep on cell type. Typ located near nucleus; in animals, often close to centrosome.
- Ea Golgi stack has two distinct faces: cis (entry; adj to ER; nearer nucleus) and trans (exit; nearer pmem).
- Outermost cisterna at ea face is conn to network of interconn membranous tubes and vesicles
For xprt thru Golgi, ______ (soluble/insoluble) proteins/mem enter ____ (cis/trans) Golgi network via xprt vesicles derived fr ER → proteins travel thru _________ in seq by means of xprt vesicles that bud fr one _______ and fuse w next → proteins exit fr ____ (cis/trans) Golgi network in xprt vesicles destined for either another endomem organelle/pmem.
For xprt thru Golgi, soluble proteins/mem enter cis Golgi network via xprt vesicles derived fr ER → proteins travel thru cisternae in seq by means of xprt vesicles that bud fr one cisterna and fuse w next → proteins exit fr trans Golgi network in xprt vesicles destined for either another endomem organelle/pmem.
Both cis/trans Golgi networks are imp for protein sorting. Describe how ea is involved.
- Enter cis Golgi → either: no ER retention signal → move onward thru stack; or, ER retention signal → returned to ER.
- Exit trans Golgi → sorted by target location: either destined for lysosomes (via endosomes) or cell surface.
Secretory proteins are released fr the cell by exocytosis via wh two pathways?
Secretory proteins are released fr the cell by exocytosis via the “constitutive” (unregulated, continuous) or regulated pathways.
Describe the regulated exocytosis pathway.
Regulated exocytosis pathway: ea specialized secretory cell produces large qty of partic prod—e.g. hormone, mucus, digestive enzymes—wh is stored in secretory vesicles → bud fr trans Golgi → accumulate near pmem → wait for EC signal that stims fusion w pmem and release of contents to EC space.
- Occurs only in cells specialized for secretion
- E.g. blood glucose: blood glucose levels ↑ → signals insulin-producing endocrine cells in pancreas to secrete insulin → insulin aggregates dissolve rapidly in blood → blood glucose ↓
Proteins destined for regulated secretion have partic surface properties. Explain.
Proteins destined for regulated secretion have partic surface props → aggregate w one/an under ionic conditions of trans Golgi (acidic pH, high Ca2+) → aggregated proteins packaged into secretory vesicles → pinch fr trans Golgi → await signal (e.g. hormone/nxmtr) to fuse w pmem.
- Selective aggregation also enables packaging of secretory proteins into secretory vesicles at concens >> concen of unaggregated protein in Golgi lumen → secretory cells promptly release large qty of protein when triggered.
- Proteins secreted by constitutive (unregulated) pathway do not aggregate → carried automatically to pmem by xprt vesicles of constitutive pathway.
Where do regulated and constitutive pathways of exocytosis diverge?
regulated and constitutive pathways of exocytosis diverge in the trans Golgi network.
Describe how phagocytic cells help defend against infection.
Phagocytic cells (incl macrophages) are widely distributed in tissues/other WBCs (e.g. neutrophils) → defend against infection by ingesting invading microorgs.
- To be taken up by macrophages/neutrophils, particles must first bind phagocytic cell surface and activate one of variety of surface receptors.
- Some receptors recog/bind antibodies, wh in turn bind microorgs/bac → induces phagocytic cell to extend sheet-like projections of pmem (pseudopods) → engulf bac and fuse at tips to form phagosome → phagosome fuses w lysosome → microbe destroyed.
Pinocytosis is carried out mainly by ________ vesicles
Pinocytosis is carried out mainly by clathrin-coated pits/vesicles.
- Clathrin-coated pits/vesicles pinch off fr pmem, trapping ECF → vesicle rapidly sheds coat and fuses w endosome → contents dissolved in ECF are delivered to endosomes.
- Fluid intake is typ balanced by fluid loss during exocytosis.
Pinocytosis is indiscriminate, i.e. endocytic vesicles simply trap any molecules that happen to be present in ECF. How, then, do cell take up specific macromolecules fr ECF?
Pinocytosis via clathrin-coated vesicles (as in animal cells) provides efficient pathway for taking up specific macromolecules fr ECF:
-
Receptor-mediated endocytosis provides selective concentrating mechanism → signif ↑ efficiency of internalization of partic macros → even minor ECF components can be taken up in large qty w/o corresponding volume of ECF.
- Macros (e.g. cholesterol) bind to complementary receptors on cell surface → enter cell as receptor–macro complex in clathrin-coated vesicles → used to synth new mem.
Cholesterol is a lipid (v insoluble in water) that binds protein to form low-density lipoprotein (LDL), wh are transported thru the bloodstream. Describe the receptor-mediated endocytosis of cholesterol.
- LDLs are secreted by liver → bind receptors located on cell surfaces.
- Receptor–LDL complexes are ingested by receptor-mediated endocytosis in clathrin-coated vesicle.
- Vesicle loses coat and fuses w endosome → LDL dissociates fr receptor (due to acidic interior of endosomes) → receptor returns to pmem for reuse via xprt vesicle whereas LDL is delivered to lysosomes.
- Hydrolytic enzymes break down LDL into free cholesterol and protein → cholesterol escapes into cytosol, whr it’s available for new mem synth.
Describe the structure and function of endosomes.
Endosomes consist of a complex set of connected mem tubes and larger vesicles; two sets of endosomes: early and late.
- Early endosomes - just beneath pmem; mature gradually into late endosomes as they fuse w ea/o or w a preexisting late endosome.
- Late endosomes - closer to nucleus; contain some lysosomal enzymes → digestion begins in endosome and continues as endosome further matures into a lysosome.
- Lysosomes - once endosome has digested most of its ingested contents → takes on dense, rounded appearance characteristic of a mature, “classical” lysosome.
Interior is kept acidic (pH 5–6) by an ATP-driven H+ pump wh pumps H+ into endosome lumen fr cytosol.
Why is the acidic environ inside endosomes critical to its role in sorting ingested molecules?
Endosome function:
- Main sorting station in inward endocytic pathway (tantamount to trans Golgi in outward secretory pathway)
-
Acidic environ is critical in sorting process → causes many (but not all) receptors to release bound cargo.
- E.g. LDL dissociates fr its receptor, but transferrin remains bound to its receptor after releasing Fe.
The fate of receptor proteins following endocytosis deps on the type of receptor. Describe three possible fates.
Fate of receptor proteins following endocytosis deps on type of receptor: recycled, degraded, or transcytosed:
- Recycled - most receptors return to same pmem fr wh they came, e.g. LDL and transferrin receptors.
- Degraded - some travel to lysosomes → degraded.
- Transcytosis - some proceed to diff domain of pmem → xfr bound cargo across cell, fr one EC space to another, e.g. apical to basolatera.
Describe the struc/func of lysosomes.
Lysosomes - membranous sacs of hydrolytic enzymes; acidic interior (pH ~5); resp for IC digestion of both EC materials and worn-out organelles.
Lysosomes contain ~40 types of hydrolytic enzymes. What happens if these enzymes escape into cytosol?
Lysosomal hydrolytic enzymes - ~40 types, incl those that degrade proteins, NAs, oligosacchs, and lipids; all optimally active in acidic lysosomal environ; nearly inactive at cytosolic pH (~7.2) → minor damage if escaped.
Describe the key components of the lysosomal membrane.
Lysosomal mem - contains xprtrs that allow final products of digestion of macros (e.g. AAs, sugars, ntides) to be xfrd to cytosol → either excreted or utilized by cell. Also contains an ATP-driven H+ pump wh pumps H+ into lysosome.
- Most lysosomal mem proteins are typ highly glycosylated (noncytosolic/lumen side) → protect proteins fr digestion by lysosomal proteases.
Describe the path of lysosomal digestive enzymes/mem proteins.
Path of lysosomal digestive enzymes/mem proteins:
- Synthd in ER → xprtd to cis Golgi and tagged w specific phosphorylated sugar group (mannose 6-phosphate).
- Moves thru Golgi to trans Golgi network, wh contains mannose 6-P receptor → sorted/packaged into xprt vesicles
- Bud off and deliver contents to lysosomes via endosomes.
There are diff paths to lysosomes, dep on source. Describe three diff paths.
Paths to lysosomes:
- EC particles are taken up into large phagosomes → fuse w lysosomes.
- ECF/macros are taken up into smaller endocytic vesicles → endosomes → lysosomes.
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Autophagy - obsolete organelles/cytosolic proteins are enclosed by double mem, creating an autophagosome → fuses w lysosome.
- AAs generated by autophagy can be recycled to allow continued protein synth.
