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74 Cards in this Set
- Front
- Back
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exocystoses
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cell excretes things out
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endocytosis
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cell takes things up
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vesicular transport
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transport vesicles bud off from one compartment and fuse with another. As they do so, they carry material as cargo from the lumen and membrane of the donor compartment.
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clathrin-coated vesicles
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transport material from plasma membrane and between endosomal and golgo compartments
has 3 large and 3 small polypeptide chains that form a triskeleton. Form a bsetketlike shape adaptor proteins form a dsicrete second layer of the coat, they bind to clathrin coat and trap proteins and capture cargo. |
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COP1 and COPII
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transport material early in the secretory pathway
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COPII
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vesicles bud from the ER
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COPI
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vesicles bud from Golgi compartments
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retromer coat
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assembles on endosomes and forms vesicles that return acid hyrolase receptors, like mannose-6-phosphate receptor to the Golgi aparatus.
only assembles when: it can bind to the cytoplasmic tails of the cargo receptors, it can interact with a curved phospholipid bilayer, it can bind to a specific phosphorylated lipid because all 3 of these things have to be met, it is a coincedence detector |
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role of dynamin in pinching off the clathrin coated vesicles
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dyamin assembles into a ring forming a bud which recriuts other proteins to the vesicle neck which destablize the lipid bilayers so taht noncytoplasmic leaflets flow together. Then the newly formed vesicle pinches off from the membrane
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Arf proteins
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responsible for COPI and caltrhin coat assembly at Golgi membranes
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Sar1 protein
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responsible for COPII coat assembly at ER membrane
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formation of a COPII coated vesicle
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Sar1 is inactive when bound to GDP. When activated by GEF, a hydrophobic tail is exposed and associates with ER membrane. Adaptor proteins sec23, and sec24 can then bind to the membrane and transmembrane receptors. Now the COPII proteins will assemble and cause the membrane to bulge. Dynamin then pinches off the vesicle
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SNAREs
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transmembrane proteins with helical domains that control vesicle transport
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V-snares
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on vesicles
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T-snares
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on target membrane
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Rab proteins are GTPases taht contribute to specificity
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there are many types. they help facilitaate and regulate the rate of vesicle docking and matching of v and t-snares
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Formation of a Rab5 domain on endosome membrane
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Rab5 specific GEF in endosome binds to Rab5 protein and exchanges GDP for GTP. Gtp binding alters conformation of Rab protein, exposing amphiphilic helix and a covalently attached lipid group which anchor Rab5-GTP to the membrane. It activates PI3- kinase
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SNAREs mediate membrane fusion
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pairing between v and t snares forces lipid bilayers into close apposition and expels water molecules from the interface. Lipid molecules in the 2 interacting leaflets of the bilayer then flow between the membranes to form a connecting stalk. Lipid of the other 2 leaflets form a new bilayer and then this rupture completes the fusion process
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interacting snares need to be pried apart before they can function again
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NSF protein catalyzes the disassembly process, it hydrolizes ATP to pry the SNAREs apart
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proteins leave the ER in what kind of coat
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COPII
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only proteins taht are properly folded can leave the ER
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If a protein is misfolded, it is held in the ER by chaperons, which will be folded or sent to the cytosol to be degraded.
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T/F
In all events involving fusion of a vescle to a target membrane, the cysosolic leaflets of the vesicle and target bilayers always fuse together, as do the leaflets that are not in contact with the cytosol |
True.
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T/F There is one strict requirement for the exit of a protein from the ER: it must be correctly folded
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True.
misfolded proteins are retained in the ER by binding to chaperone proteins like BiP. |
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T/F ALl of the gylocoproteins and glycolpipids in intracellular membranes have their oligosaccharide chains facing the lumenal side
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True
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T/F during transcytosis, vesicles that form from coated pits on the apical surface fuse with the plasma membrane on the basolateral surface, and in that way transport molecules across the epithelium
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Flase. vesicles first fuse with endosomes
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In a nondividng cell such as a liver cell, why must the flow of membrane between compartments be balanced, so that the retrival pathways match the outward flow?
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if not balanced, some compartments would grow and some would shrink. Keeping all the compartments about the same time is essential for proper functionining of a liver cell. In a growing cell, all of the compartments must double in size to generate 2 daugther cells. so there will be an imbalance in favor of the outward flow, which will be supported by the new membrane synthesis equal to the sum total of all the membranse
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Yeast and many other organisms make up a single type of clathrin, heavy chain and a single type light chain; thus the make a single clathrin coat. How is it then, that a single coat can be used for 3 different transport pathways that each involve different specialized cargo proteins?
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The specificty comes from the adaptor proteins that link the clathrin to the transmembrane receptors for specific cargo proteins. The different adaptor proteins allow different cargo receptors, so different cargo proteins, to be transported along specific pathways.
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How can it possibly be true that complementary pairs of specific SNARES uniquely mark vesciles adn their target membranes? After vesicle fusion, the target membrane will contain a mix of V and t snares. Initially these are tightly bound but NSF can pry them apart. What do you suppose prevents target membranes from accumulating a poplation of V snares equal to or greater than t snares
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There are always V-snares in the target membrane. After fusion, the v-snares form inactive compelxes with t-snares. Once NSF pries them apart, V-snares may be kept active by binding to inhibitory proteins. Build up of v-snares in the target membrane beyond a minimal population is thought to be prevented by active retrival pathways that incorporate v-snares into vesicles for redilivery to the orignial donor membrane
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Viruses are the ultimate scavangers, a necessary consequence of their small genomes. Whereever possible, they make use of the cells machinery to accomplish the steps involved in their own reproduction. Many have membrane coverings. these so called enveloped viruses gain access to the cytosol by fusing with a cell membrane. Why do you suppose each of these viruses encodes its own fusion protein rather than making use of a cells SNARES?
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In a cell, SNares are bound to membrane cytosolic surface and function by juxtapositioning the cytosilic surfaces of the 2 membranes to be fused. Viruses, must fuse with a cell membrane by bringing together its external surface with a membranes external surface. So enveloped viruses cannot make use of cells snares because they are located on the wrong side of the membrane.
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SNAREs exist as complementary partners that carry out membrane functions between appropriate vesicles and their target membranes..long question..
What do these data say about the requirements for V-snares and t-snares in the fusion of vacuolar vesicles? Does it matter which snare is on which vesicle |
To generate maximal alkaline phosphate activity, vesicles from each strand must carry both V and t snares. If either vesicle is lacking v or t, phosphatase activity is reduced to 30-60% if the max. If both vesicles are missing V or T, phosphatase activity is super low, like it is if one vesicle is missing both SNARES. So for a good level of fusion, complementary snares must be present on the vesicle. It doesnt matter which snare is on a vesicle as long as vesicles from strain b carry a complementary snare;
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If you were to remove the ER retrival signal from protein disulfide isomerase, which is normally a soluble resident of the ER lumen, where would you expect the modified PDI to be located?
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Outside of the cell. If PDI were missing the ER retrival signal, its flow out of the ER to the Golgi would not be countered by capture and return to the ER. it would leave the golgi by the default pathway and would not be retained anywheere else because it has no signals for localization
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The KDEL receptor must shuttle back and forth between the ER and the Golgi apparatus to accomplish its task of ensuring that soluble ER proteins are retained in the lumen. In which compartments does the KDEL receptor binds its ligands more tightly? More weakly? What is thought to be the basis for its different binding affinities in the 2 compartments. In which compartment would you have the highest concentration of KDEL receptor?
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More tightly- Golgi aparatus where it captures proteins that escaped into the ER so it can return them.
More loosely- in the ER so that those captured proteins in the Golgi can be released when they return to ER. Basis for different binding is the slight different in pH. The lumen of the golgi apparatus is slightly more acidic than the neutral ER. Since KDEl captures proteins that have escaped from ER, design the system so that recoptrs are found in highest concentration in the Golgi apparatus. KDEL does not have a classic ER retrval signal but it has a conditional retrival singal, upon binding to an ER protein in the golgi, its conformation is altered so taht a binding site for COPI subunites is exposed. |
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How does a low pH of lysosomes protect the rest of the cell from lysosomal enzymes in case the lysosome breaks?
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lysosomal enzymes are all acid hydrolases which work best at low pH(about 5). If a lysosome breaks, the acid hydrolases would be in a higher pH(7.2), the cytosolic pH and therefore it wouldnt do much damage.
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Melanosomes are specialized lysosomes that store pigments for eventual release by exocytosis. Various cells such as skin and hair cells then take up the pigment....How might the loss of AP3 cause a defect in melanosomes
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Adapter proteins mediate the incorporation of specific cargo proteins into a clathrin coated vesicles by linking the clathrin coat to specific cargo receptors. Because melanosomes are specalized lysosomes, the defect in AP3 affects the trans golgi network which involves calthrin coated vesicles. AP3 localizes to the trans golgi network.
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Patients with Hunters syndrome or Hurlers syndrome rarely live beyond their teens. A) what do you suupose the corrective factors are? B) Why do you suppose the treatments with protease periodate and alkaline inactivate the corrective factors? C) would you expect a similar sort of correction scheme to work for mutant cytosolic enzymes
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A. Corrective factors are lysosomal enzymes themselves. Hurlers cells provide the missing enzyme from Hunters cells and vice versa. These enzumes are present in the medium because of the inefficiency of the sorting process. Since they carry M6P, which should direct them to lysosome, they escaped capture and were secreted. THey are taken into cells and delivered to lysosomes by receptor mediated endocytosis which operates due to M6P receptors on the cell surface. The degradative enzyme are eventually delivered to lysosomes. Lysosomes are the normal site of action, so defect is corrected
B) protease treatment destroys the lysosomal enzymes. Periodate treatment and alkaline treatment remove M6P singal that is required for binding to rececptor and it prevents the enzyme from entering the cell C) No. External proteins usually dont cross the membrane so even when the are taken up in the cell, they stay in the lumen. Also foreing proteins are usually degraded. |
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transport vesicle
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general term for a membrane enclosed container that moves
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Rab protein
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any of a large family of monermic GTPases present in the plasma membrane and organelle membrane that confer specificity
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adaptor protein
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a protein that mediates binding between the clathrin coat and transmembrane proteins including transmembrane cargo receptors
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Dynamin
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cytosilic GTPase taht binds to the neck of a clathrin coated vesicle and helps it to pinch off from the membrane
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NSF
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the protein that catalyzes the dissasemly of helical domains of paired snare proteins
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clathrin coated vesicle
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coated vesicle that transports material from the plasma membrane and between endosomal and golgi compartments
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Arf protein
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the coat recruitment GTPase responsible for both COPI and clathrin assembly
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Rab effector
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protein that facilitates vesicle transport, docking and membrane fusion
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SNARE protein
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general term for a member of the large family of proteins that catalyze the membrane fusion reactions in membrane transport
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retromer
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multiprotein complex that assembles on endosomal membranes only when it can bind the cytoplasmic tail of a receptor
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coated vesicle
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general term for transport vesicle that carries a distinctive cage of protein covering its surface
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sar1 protein
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coat recruitment GTPase responsible for COPII assmebly at the ER membrane
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at least 3 different coats form around transport vesicles. What 2 principal functions do these coats have in common?
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1) concentrate specific membrane proteins
2) bend membrane into a sphere |
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discuss the following analogy: cargo receptors competitng to be transported..
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cargo receptors can only be incorporated to a coated vesicle if they can bind to adaptor proteins(have ticket) which allows them to enter the coated pit and be incorporated into a vesicle(cable car). Cargo receptors(skiers) are mixed together without any guarunteed travel companion but all get to the next compartment (station)
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Clathrin coated vesicles bud from eucaryotic plasma membrane fragments when adaptor proteins are added. What would you expect to observe if..
A. Adaptor proteins were ommitted B. Clathrin ommited C. Dynamin was ommited D. Procaryotic membrane fragments were used |
A. clathrin coated vesicles cannot assemble
B.Without clathrin, adaptor proteins still bind to receptors in the membrane, but no clathrin coat can form. Thus, no clathrin-coated pits or vesicles will form. C. In the absence of dynamin, clathrin-coated pits can form and proceed toward vesicle formation, but the last step—membrane fusion—is blocked without dynamin. As a result, deeply invaginated coated pits will be observed in the absence of dynamin D.A procaryotic cell does not contain any receptors with appropriate cytosolic tails that could mediate the binding of adaptor proteins. Therefore, no clathrin-coated vesicles will form. |
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imagine that Arf1 protein was mutated so that it could not hydolize GTP. Would you expect COPI coated vesicles to form normally?
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Arf1 would exist in a cell as Arf1–GTP. Since Arf1–GTP promotes assembly of COPI-coated vesi- cles, you would expect such vesicles to form readily, but they might not form at the right place in the cell. Normally, Arf1 is delivered specifically to the Golgi membrane by a Golgi-bound Arf1–GEF, which converts the cytosolic Arf1–GDP to Arf1–GTP, exposing a fatty acid tail that allows it to bind to the membrane. The mutant Arf1, which would always have GTP bound and its fatty acid tail exposed, might bind inappropriately to other cell membranes, thus promoting COPI-coated vesicle formation at inap- propriate places in a cell.
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When the fungal metabolite brefeldin A is added to cells.. how do you think brefeldin A blocks formation of COP1 coated vesicles
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These observations indicate that brefeldin A blocks COPI-coated vesicle for- mation by interfering with the exchange of GTP for GDP, which is essential for Arf to bind to the Golgi membrane and initiate formation of coated vesi- cles.
Observation 1 shows that brefeldin A does not block coated vesicle forma- tion if Arf is first locked into its active form by GTPgS. Thus, the assembly of the COPI coats and formation of vesicles are not affected by brefeldin A, if the active membrane-bound form of Arf is present. Observation 2 shows that a protein in the Golgi membrane—a GEF—cat- alyzes the exchange of GTP for GDP. This exchange reaction is blocked by brefeldin A. Observation 2, by itself, does not distinguish whether the effect of brefeldin A is on the GEF, or on Arf: binding of brefeldin A to either protein could interfere with the exchange reaction. In fact, recent experiments show that brefeldin A binds to the complex of Arf and GEF, locking Arf into a non- productive GDP-bound conformation. |
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cisternal progression model
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hyoithesis that new cisternae form continouly at the cis face of the golgi and then migrate through the stack as they mature
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cis-face
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side of the golgi stack at which material enters the organelle
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trans golgi network
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meshwork of interconnected cisternae and tubules on the side of the golgi
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T/F tall of the glycoproteins and glycolipids in intracellular membranes have their oligosaccharide chains facing the lumenal side, and all those in the plasma membrane have their oligosaccorides chains facing the outside of the cell
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true
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t/f the golgi apparatus confers the heaviest glycostation of all on proteoglycan pore proteins
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true
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Calnexin caltriculin an dHMG Coa reductase. which is soluble and which is trasnmembrane
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Calnexin and HMG CoA reductase are transmembrane proteins and calreti- culin is soluble. Know this by looking at C-term. Calticulin has KDEL, the ER retreival signal for soluble proteins, at its C-term.
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3 functions of protein carbhydrates
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1)promote protein folding by making the interme- diates more soluble and mediating their binding to chaperones. (
2)serve as a recognition marker for transport from the ER and for protein sorting in the trans Golgi network. 3)Oligosaccharides on pro- teins provide protection against proteases. |
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T/F lysosomal membranes contain a proton pump that utilizes the engery of ATP hydrolysis
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False
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t/f late endosomes are convereted to mature lysosomes by the loss of distinct endosomal membrane proteins and a further decrease in initial pH
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true
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t/f if cells were treated with a weak base such as ammonia, which raises pH, m6P receptors would be expected to accumulate in the golgi because they could not bind to the lysosomal enzymes
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false
if pH were raised, M6P could not release their bound enzymes and would be trapped in the late endosome |
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how many layers separate the matric of mitochondrion from cytosol?
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4
ER membrane, ER lumen, ER membrane, cytosol, outer mitochondrial membrane, intermembrane space, inner mitochondrial membrane, and matrix. |
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m6p
a)what would happen if pH were raised to 6.6 ph? b) lowered to pH 6? |
a) they would bind hydrolases normally and transport them to late endosomes but would not be able to release the hydrolases and so it can not be recycled back to trans golgi network
b)he M6P receptor would not bind to the lysosomal hydrolases, and thus could not deliver them to late endosomes via the principal transport pathway. Under these condi- tions, the hydrolases would exit the cell via the default pathway |
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t/f any particle that is bound to the surface of a phagocute will be ingested by phagocytosis
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false
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t/f like the LDL receptor, most of more than the 25 different receptors known participate only after they have bound specific ligands
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flase
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t/f all the molecules that enter early endosomes ultimatly reach late endosomes
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false
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t/f during transcytosis, vesicles that form from coated pits on the apical surface fuse with the plasma membrane on the basolateral surface
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false
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what might you predict would be a characteristic of the structure of transmembrane proteins taht collect in caveolae?
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since lipids rafts are thicker than other areas of the plasma membrane, the membranes inside caveolae, which form from lipid rafts, are presumably also thicker. Thus, transmembrane proteins that collect passively in caveolae might be expected to have longer transmembrane segments than normal.
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t/f when a foregin gene encoding a secretory protein is introduced into a secretory cell that normally does not make the protein, the alien protein is not packeaged into secertory vesicles
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false
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t/f once a secretory vesicle is properly positioned beneath the plasma membrane, it will immediatly fuse with the membrane
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false
waits until the cell receives an appropriate signa |
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if you replace Ran protein in the cell with Ran protein that has an amino acid change locking Ran in a GDP bound state, how will this effect nuclear import and export
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nuclear export would stop working because it needs the conversion of Ran GDP to RanGTP to either release proteins into nucleus or to release cargo into cytosol
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which protein functions as a coat recriutment GTPase during vesicle formation on ER membrane?
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SAR1
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how does sar1 recruit protein coat?
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Sar1 has GDP bound to it and converts it GTP through Sar GEF protein. This change will now signal for COPII proteins to bind
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