Term
| What are the 2 possible fates of a protein synthesized in the ER? |
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Definition
1. retained in the ER
2. exits the ER via ERES |
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Term
| What are ERES? Where are they found? |
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Definition
Endplasmic Reticulum Exit Sites
-subdomains of the ER, usually next to cis face of a Golgi complex |
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Term
| What is/are the role(s) of ERES machinery? |
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Definition
-formation of transport vesicles destined for Golgi (budding)
-proper packaging of vesicles with correct lumenal & membrane proteins and lipids |
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Term
| Describe the morphology of vesicles formed at the ERES |
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Definition
-small, 20-50mm
-cytosolic surface of membrane has a layer of soluble coat proteins (COP) |
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Term
| What are COP? What are the main functions of COP? |
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Definition
Coat Proteins
1. identify & concentrate specific proteins/lipids that will be incorporated into a vesicle
2. mediates ERES membrane & vesicle membrane curvature |
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Term
| What are the major classes of coat proteins, and their roles? |
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Definition
COP II
-anterograde transport
COP I
retrogade transport (Golgi to ER & backwards within the Golgi)
Clathrin
-vesicles with a clathrin coat move from Golgi or PM to endosomes |
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Term
| Describe COPII-coated vesicle assembly at the ERES |
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Definition
1. Sar1-GDP (CopII component) is recruited to cytosolic surface of ERES, binds a GEF, forms Sar1-GTP
2. Sar1-GTP integrates into outer leaflet at ERES, initiates membrane curvature
3. Sec23 & Sec24 are recruited to cytosolic surface of ERES membrane
-further curvature
-Sec24 concentrates select proteins within growing bud
4. Sec13 & Sec31 are recruited to surface, form outer scaffolding of coat
5. CopII coat fully assembled, vesicle pinches off |
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Term
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Definition
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Term
| What happens to the COP oat of a vesicle prior to fusion with its destination membrane? |
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Definition
COP coat disassembles
- Sar-GTP converted back to Sar-GDP, released into cytosol along with other COP |
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Term
| What is function of Sec23 & Sec24? |
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Definition
| form a dimer to promote further bending of the ERES membrane |
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Term
| To which proteins does Sec24 bind? |
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Definition
Binds to cytosolic domains of specific integral proteins
-cargo proteins destined to exit the ER
-cargo receptor proteins which will bind lumenal cargo proteins
-membrane receptor proteins which assist in trafficking & docking of the vesicle |
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Term
| What face of the Golgi is adjacent to ERES? |
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Definition
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Term
| What mediates the movement of vesicles through the cytosol? |
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Definition
| cytoskeleal network & molecular motor proteins such as kinesin |
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Term
| Summarize the steps of targeting & fusion of a vesicle from the ER to CGN |
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Definition
1. Rab proteins mediate recognition of incoming vesicle & recipient membranes
2. Vesicle is tethered to recipient membrane
3. SNARE proteins mediate docking of vesicle at recipient membrane
4. Vesicle & recipient membranes fuse |
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Term
| What are Rab proteins, where are they found, what do they do? |
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Definition
-lipid anchored membrane proteins
-found on all transport vesicles & recipient membranes
-convey vesicle targeting specificity, involved in recognition & tethering |
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Term
| What is required for association of a Rab protein with a membrane? |
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Definition
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Term
| Describe the process of tethering a recipient vesicle to the CGN recipient membrane |
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Definition
1. GTP-Rabs recruit tethering proteins from cytosol to surface of vesicle & recipient membranes
2. Tethering proteins on opposing membranes interact to form a bridge, bringing the two membranes close together |
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Term
| What are SNARE proteins, where are they located and what do they do? |
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Definition
-integral membrane-bound proteins
-located on all transport vesicle & recipient membranes
-convey vesicle targeting specificity, involved in docking |
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Term
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Definition
| a domain that extends from a membrane surface into the cytosol, mediates SNARE-SNARE protein binding |
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Term
| What are the 2 main classes of SNARE proteins? |
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Definition
v-SNARES: found on transport vesicle membranes, incorporated during budding at the ERES
t-SNARES: found on target membranes |
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Term
| Describe what happens during fusion of a vesicle membrane with a target membrane |
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Definition
-vesicle membrane proteins move laterally into recipient membrane
-soluble cargo proteins are released into lumen |
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Term
| What happens to vesicle-specific proteins or proteins that escaped from the ER and end up in the CGN? |
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Definition
| ER retrieval signals ensure they are returned back to the Golgi via retrograde transport |
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Term
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Definition
| an ER retrieval signal on the C-terminus of soluble resident ER proteins |
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Term
| Describe the action of a KDEL receptor/retrograde transport |
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Definition
1. KDEL receptor recognizes escaped proteins in CGN lumen by their KDEL sequence
2. lumenal side of receptor binds to KDEL sequence of the protein, cytosolic side binds to COPI components
3. COPI mediates formation of a vesicle on at the CGN
4. Vesicle returns to the ER, KDEL receptor releases the ER protein
5. KDEL receptor is returned to the CGN via COPII vesicles |
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Term
| What kind of retrieval signal is found in ER membrane proteins? Soluble ER proteins? |
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Definition
ER membrane proteins: KDxx
Soluble ER proteins: KDEL |
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Term
| What kinds of targeting events/sequences would bring a KDEL receptor to its destination post-synthesis? |
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Definition
-signal sequence binding to SRP receptor
-Sec24 binding to cytosolic domain of receptor
-KKxx/KDEL forming part of the cytosolic domain for retrieval |
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Term
| Describe the polarity/orientation of the Golgi apparatus |
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Definition
cis Golgi network (medial to ER)
cis cisternae
medial cisternae
trans cistarnae
trans Golgi network |
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Term
True or False
Cisternae are biochemically unique |
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Definition
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Term
| Which have multiple Golgi complexes, mammalian or plant cells? |
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Definition
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Term
| Osmium tetroxide is exclusive to what region of the Golgi apparatus? |
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Definition
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Term
| Mannosidase II is exclusive to what region of the Golgi apparatus? |
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Definition
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Term
| Nucleoside diphosphatase is exclusive to what region of the Golgi apparatus? |
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Definition
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Term
| What mediates the organization of the Golgi complex? |
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Definition
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Term
| What is the Golgi matrix? |
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Definition
| various peripheral & membrane proteins which mediate organization of the Golgi & link the Golgi to the cytoskeleton |
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Term
| What are GRASPs? What do they do? |
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Definition
Golgi ReAssembly and Stacking Proteins
-serve as tethering proteins to link different Golgi subcompartments together
-facilitate movement of Golgi along microfilaments throughout the cell |
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Term
| What would happen if transcription of GrasP55/65 was reduced or halted? |
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Definition
| the Golgi complex would likely disassemble, as these are what links the Golgi subcompartments together |
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Term
| Compare the structure of the cis/trans golgi networks to the cisternae |
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Definition
CGN & TGN are a combination of tubules & vesicles
cisternae consist of large, flattened sacs |
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Term
| What is the function of the GGN? |
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Definition
Acts as a sorting station
-receives COPII vesicles from ERES
-assembles COPI vesicles for retrograde transport, or anterograde further into Golgi |
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Term
| What is the function of the TGN? |
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Definition
Acts as a sorting station
-anterograde transport to enosome/lyosome/PM
-retrograde transport to trans cisternae |
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Term
| Where does Golgi metabolism occur? |
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Definition
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Term
| Where are hemicellulose & cellulose produced? |
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Definition
| in the cisternae of the Golgi apparatus |
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Term
| What are the general functions of the Golgi? |
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Definition
-synthesis of complex polysaccharides
-modification of proteins & lipids
-transport & sorting of proteins |
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Term
| Where are glycosyltransferase enzymes found? |
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Definition
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Term
| What is the difference between N-linked and O-linked oligosaccharides? |
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Definition
N-linked: synthesis begins in ER, continues in Golgi
O-linked: synthesis & modification are entirely in Golgi |
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Term
| What does alpha-mannosidase I do? |
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Definition
| removes 3 mannose sugars from the core oligosaccharide |
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Term
| Describe how modifications of proteins in the Golgi can contribute to protein targeting by the addition of a mannose-6-phosphate group |
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Definition
1. Lysosomal protein arrives in CGN from RER
2. cis cisternae: N-acetylglucosamine-1-phosphate is transferred to a mannose residue on the protein
3. medial Golgi: N-acetylglucosamine group is cleaved, protein now has a mannose-6-phosphate group
4. TGN: mannose-6-phosphate receptors (MPRs) recognize the protein, package into vesicles destined for the lysosome |
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Term
| Where will cargo proteins without an M6P group be sent from the TGN? What pathway will they follow? |
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Definition
| to the plasma membrane or other organelles via secretory or regulatory pathway |
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Term
True or False
Golgi subcompartments are continuously maturing, changing chemical composition and moving from the cis to the trans side of the Golgi complex |
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Definition
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Term
| Where does N-linked glycosylation occur? |
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Definition
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Term
| Describe the cisternal progression/maturation model of the Golgi network |
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Definition
-COPII vesicles arrive at cis side of Golgi complex, fuse together to form a new CGN
-new CGN matures into cis cisternae, medial, then trans, changing chemical position in the process
-TGN disperses into various types of vesicles
-COP I vesicles transport resident Golgo enzymes 'back' to where they belong |
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Term
| What types of vesicles might be formed the the TGN? |
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Definition
Clathrin-coated vesicles
-transport cargo to endosomes/lysosomes
Secretory vesicles
-transport cargo to PM & extracellular space
Secretory granules
-fuse with PM & release cargo into extracellular space |
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Term
| Where are M6P-tagged proteins destined to end up? |
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Definition
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Term
| Where are lysosomal proteins N-glycosylated? |
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Definition
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Term
| What happens when glycosylated lysosomal proteins reach the cis Golgi cisternae? |
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Definition
| -2 mannose residues are phosphorylated...mannose-6-phosphate (M6P) |
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Term
| What is the function of mannose-6-phosphate receptors (MPR)? |
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Definition
| mediate recruitment of lysosomal proteins into nascent clathrin-coated vesicles |
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Term
| What is the function of GGA proteins? |
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Definition
| they act as linkers during clathrin-coated vesicle assembly |
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Term
| What do the 2 domains of MPR bind to? |
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Definition
Lumenal domain binds to M6P groups on proteins destined for lysosomes
Cytosolic domain binds to GGA adaptor coat proteins |
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Term
| How are GGA adaptor proteins recruited to the surface of the TGN? |
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Definition
| Arf 1-GTP is bound to the cytosolic side of TGN membrane, & also to GGA proteins |
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Term
| What is the function of Arf1-GTP/GDP? |
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Definition
-mediates recruitment of GGA adaptor proteins
-initiates membrane curvature of TGN membrane during formation of clathrin-coated vesicles |
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Term
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Definition
| A molecule of clathrin, consisting of 3 light & 3 heavy chain polypeptides |
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Term
| Desribe the processs of formation of the inner layer of a clathrin-coated vesicle |
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Definition
1. MPR binds M6P tagged protein on lumenal side, GGA protein on cytosolic side
2. Arf1 binds to TGN membrane, mediates GGA recruitment & initiates membrane curvature
3. GGA will bind to the rest of the vesicle coat (clathrin) |
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Term
| Desribe the processs of formation of the outer layer of a clathrin-coated vesicle |
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Definition
1. Clathrin triskelions self-assemble to form hexagons
2. Triskelions proceed to form pentagons, driving further membrane curvature |
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Term
| Describe the "pinching off" process in clathrin-coated vesicle assembly at the TGN |
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Definition
1. Dynamin is recruited from cytosol to the stalk connected the clathrin-coated bud to the TGN membrane
2. Dynamin polymerize to form a ring surrounding the stalk
3. GTP hydrolysis changes conformation of dynamin ring, causes vesicle to twist & pinch off
4. Clathrin coat disassembles, Arf1-GTP converted back to Arf1-GDP & released with GGA & triskelions into cytosol |
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Term
| What is the effect of gamma-GTP on dynamin ring polymerization? |
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Definition
| causes polymerization to continue, extending the stalk between the clathrin bud & TGN membrane rather than pinching it off |
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Term
| How are the contents of a nascent vesicle destined for the lysosome released into the lumen of a late endosome? |
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Definition
-vesicle targets to & fuses with late endosome (Rabs & SNARES!)
-acidic interior of late endosome causes MPRs to dissociate from their proteins
-lysosomal proteins are partitioned to a specific region of the late endosome, which will eventually detach and fuse with lysosome |
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Term
| What happens to MPRs after they are released from their lysosomal cargo proteins in the late endosome? |
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Definition
| they are recycled back to the TGN for another round of trafficking |
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