Intracellular Transport

Question 1

Protein transfer to all organelles except ___ is post-translational and follows synthesis on ___ ribosomes, mediated by organelle-specific ___ ____ sequences.

Proteins destined to be in ___ and ____ do not have a ____.

Answer 1

Protein transfer to all organelles except ER is post-translational and follows synthesis on FREE ribosomes, mediated by organelle-specific SIGNAL AA sequences.

Proteins destined to be in CYTOPLASM and CYTOSKELETON do not have a SIGNAL SEQUENCE.

Question 2

Specific GTP binding proteins involved in cell trafficking:

1. Release of nuclear proteins from receptor used for import ___
2. Formation of transport vesicles ____, ____
3. Transport vesicle recognition by target organelles ____

Answer 2

Specific GTP binding proteins involved in cell trafficking:

1. Release of nuclear proteins from receptor used for import RAN GTPASE (FOUND ONLY IN NUCLEUS)
2. Formation of transport vesicles ARF, SAR-1
3. Transport vesicle recognition by target organelles RAB GTPASE (FOUND ONLY IN CYTOPLASM)

Question 3

How do nuclear proteins get to the nucleus?

1. cargo protein with ____ binds to cytosolic receptor ___
2. The _ subunit of ____ binds to ___ of the cargo protein. The _ subunit binds to ____.
3. ____ _ subunit dissociated during translocation and remains in cytoplasm
4. _ subunit is transported through nuclear pore complex in association with cargo
5. Inside nucleus, GTP bound ___ induces the separation of the cargo protein from _ subunit. An export cycle involved ____, which induced binding of cargo protein to ____ for cytoplasmic transport. Note that ___ is the driving force for both protein import and export.

Answer 3

How do nuclear proteins get to the nucleus?

1. cargo protein with NLS binds to cytosolic receptor IMPORTIN
2. The A subunit of IMPORTIN binds to NLS of the cargo protein. The B subunit binds to NPC.
3. B subunit dissociated during translocation and remains in cytoplasm
4. A subunit is transported through nuclear pore complex in association with cargo
5. Inside nucleus, GTP bound RAN induces the separation of the cargo protein from A subunit. An export cycle involved RANGTP, which induced binding of cargo protein to EXPORTIN for cytoplasmic transport. Note that GTP is the driving force for both protein import and export.

Question 4

In Zellweger Syndrome, ___ ____ proteins are missing _____ (____-___-____ at _____).

Symptoms: ____, _____

Answer 4

In Zellweger Syndrome, PEROXISOMAL ENZYME proteins are missing SIGNAL (SER-LYS-LEU at C-TERMINUS).

Symptoms: DEATH, EMPTY PEROXISOMES

Question 5

Secretory protein synthesis begins in ____ and finishes in _____.

____-terminus of secretory proteins have _______ that binds _____ when ____ emerges from _____ ribosome.

When _____ receptor in ____ is ___-bound, it can bind to ____-____ complex. When _____ is hydrolyzed on both _____ and ____, _____ goes away and protien synthesis resumes.

Eventually ____-terminal ____ is cleaved, and protein will get _____.

Answer 5

Secretory protein synthesis begins in CYTOPLASM and finishes in RER.

N-terminus of secretory proteins have SIGNAL PEPTIDE SEQUENCE that binds SRP when SIGNAL PEPTIDE emerges from CYTOSOLIC ribosome.

When SRP receptor in RER is GTP-bound, it can bind to SRP-RIBOSOME complex. When GTP is hydrolyzed on both RECEPTOR and SRP, SRP goes away and protien synthesis resumes.

Eventually N-terminal SEQUENCE is cleaved, and protein will get FOLDED.

Question 6

The several protein processing steps during RER protein synthesis (this all occurs on _____)

• _______ removal
• _____ of lys and pro
• _____ bond formation
• folding via ______ proteins
• addition of _____ used to assess proper _____

Answer 6

The several protein processing steps during RER protein synthesis (this all occurs on MEMBRANE)

• SIGNAL PEPTIDE removal
• HYDROXYLATION of lys and pro
• DISULFIDE bond formation
• folding via CHAPERONE proteins
• addition of SUGARS used to assess proper FOLDING

Question 7

Steps in glycosylation:

• ________ oligosaccharide with 9 _____ added co-translationally from a membrane ________ to specific residues on protein chain, usually ___.
• addition of carbohydrates never on _____ side.
• oligosaccharide is _____ by ________ as protein is transported through cell (usually modified in _____).
• the __-glycosylation modifications are used to assess ______.

Answer 7

Steps in glycosylation:

• PRE-FORMED oligosaccharide with 9 MANNOSES added co-translationally from a membrane LIPID DONOR to specific residues on protein chain, usually ASN.
• addition of carbohydrates never on CYTOPLASMIC side.
• oligosaccharide is MODIFIED by VESICLE-SPECIFIC ENZYMES as protein is transported through cell (usually modified in GOLGI).
• the ER-glycosylation modifications are used to assess PROPER PROTEIN FOLDING.

Question 8

Assembly of COPI/II coats are regulated by _______ that cycle between active and inactive states.

1. Inactive state- ____-bound
2. _____ protein releases ___ and allows ____ to bind, causing conformational change–> active state.
3. Activation of ____ hydrolyzes ___ and returns to inactive state.

The ____ proteins involved in COPI is ___
The ____ proteins involved in COPII is ___

Answer 8

Assembly of COPI/II coats are regulated by GTP-BPS that cycle between active and inactive states.

1. Inactive state- GDP-bound
2. GEF protein releases GDP and allows GTP to bind, causing conformational change–> active state.
3. Activation of GTPase hydrolyzes ATP and returns to inactive state.

The GTPBPS proteins involved in COPI is ARF
The GTPBS proteins involved in COPII is SAR-1

Question 9

Formation of COP Vesicles:

• Free-floating ____ GTP binding proteins are bound by ____. It’s ____ is unexposed.
• When ____ approaches membrane, it is acted upon by membrane-associated ____. ____ is now ____-bound, and the ____ is exposed, allowing for insertion into membrane.
• Other subunits bind and ____ begins to form. These subunits can bind ____/____ receptors. This is how ____ get selected for transport.
• ____ layer of subunits drives final budding.

Answer 9

Formation of COP Vesicles:

• Free-floating SAR1 GTP binding proteins are bound by GDP. It’s FA TAIL is unexposed.
• When SAR1 approaches membrane, it is acted upon by membrane-associated GEF. SAR1 is now GTP-bound, and the FA-TAIL is exposed, allowing for insertion into membrane.
• Other subunits bind and VESICLE begins to form. These subunits can bind CARGO/CARGO receptors. This is how PROTEINS get selected for transport.
• OUTER layer of subunits drives final budding.

Question 10

Directionality of Coated Vesicles:

COPII: ____ to _____

COPI: ______

Clathirin: _____ to _____, ____ to ____, and _____ to _____.

Answer 10

Directionality of Coated Vesicles:

COPII: ER to CIS GOLGI

COPI: RETROGRADE

Clathirin: CELL SURFACE to EARLY ENDOSOME, TGN to LATE ENDOSOME, and LATE ENDOSOME to TGN.

Question 11

V and T-SNARES are _____ membrane proteins that mediate vesicle-target ______ and ______.

• If correct fit, initiation of ______ wrapping until _____ _____.
• _____binding proteins called _____ also involved in vesicle targeting by binding ______ when ____ is bound.
• Botulinum and tetanus toxins cleave _____, thus blocking ______.

SNARE hypothesis predicted that _____ ______ SNARE membrane proteins are used for recognition in _____ transport steps

Answer 11

V and T-SNARES are COMPLEMENTARY membrane proteins that mediate vesicle-target RECOGNITION and FUSION.

• If correct fit, initiation of COILED-COIL wrapping until MEMBRANES FUSE.
• GTP-binding proteins called RAB also involved in vesicle targeting by binding EFFECTORS when GTP is bound.
• Botulinum and tetanus toxins cleave SNARES, thus blocking VESICLE FUSION.

SNARE hypothesis predicted that DIFFERENT SETS OF COMPLEMENTARY SNARE membrane proteins are used for recognition in DIFFERENT transport steps

Question 12

Bringing it all together: Formation and Fusion of Vesicles

1. _____ binding protein, ____ coat, SNARE, and ____ recruitment
2. Coat completed with ____ layer coat proteins that ____ membrane.
   3,4. _____ from surface and ______ to expose SNARES; ____ gets recycled.
3. ____ may mediate initial attachment, likely recognized in part by ___
   6,7. Docking via ______ interaction, then fusion

Answer 12

Bringing it all together: Formation and Fusion of Vesicles

1. GTP binding protein, INNER coat, SNARE, and CARGO recruitment
2. Coat completed with OUTER layer coat proteins that DEFORM membrane.
   3,4. SCISSION from surface and UNCOATING to expose SNARES; COAT gets recycled.
3. TETHER may mediate initial attachment, likely recognized in part by RAB
   6,7. Docking via SNARE interaction, then fusion

Question 13

Retrieval of mis-routed vesicles via ______ by reading _____ sequence on escaped proteins associated with _____ vesicles.

Answer 13

Retrieval of mis-routed vesicles via COPI by reading KDEL sequence on escaped proteins associated with COPII vesicles.

Question 14

______ which begun in ER continues in ______. Then, proteins reach the ____ which is the sorting station in biosynthetic pathway.

Answer 14

SUGAR MODIFICATION which begun in ER continues in GOLGI. Then, proteins reach the TGN which is the sorting station in biosynthetic pathway.

Question 15

4 Sorting Pathways from TGN

1. Signal-mediated diversion to _____
2. Signal-mediated diversion to ______(for ______ ____)
3. ________ _____ ______
4. To ______

Answer 15

4 Sorting Pathways from TGN

1. Signal-mediated diversion to LYSOSOMES
2. Signal-mediated diversion to SECRETORY VESICLES(for REGULATED SECRETION)
3. CONSTITUTIVE SECRETORY PATHWAY
4. To MEMBRANE

Question 16

_____ are usually processed after they leave TGN

Answer 16

PROHORMONES are usually processed after they leave TGN

Question 17

Lysosomal proteins are marked by the addition of ____ to a _____ residue. The modification is recognized by specific ____ receptors in the _____, packaged into ____ vesicles, and delivered to _____.

In _____ disease, _____ modificaiton of ___ on lysosomal proteins does not occur because the _____ is mutated.

Since ______ modified lysosomal proteins are not produced, the lysosomal proteins cannot be recognized by ___ receptor, and are not packaged into vesicles bound for lysosomes. Instead, all of these lysosomal enzymes are _____.

Answer 17

Lysosomal proteins are marked by the addition of PHOSPHATE to a MANNOSE residue. The modification is recognized by specific M6P receptors in the TGN, packaged into CLATHRIN vesicles, and delivered to LYSOSOMES.

In I-CELL disease, M6P modificaiton of MANNOSE on lysosomal proteins does not occur because the PHOSPHOTRANSFERASE is mutated.

Since M6P modified lysosomal proteins are not produced, the lysosomal proteins cannot be recognized by M6P receptor, and are not packaged into vesicles bound for lysosomes. Instead, all of these lysosomal enzymes are SECRETED.

Question 18

_______ is the sorting system in the _____ pathway, just as the TGN is the sorting pathway in the secretory pathway

Answer 18

EARLY ENDOSOME is the sorting system in the ENDOCYTIC pathway, just as the TGN is the sorting pathway in the secretory pathway

Question 19

______ _____ is the sorting system in the ____ pathway just as the TGN is the sorting system in the _____ pathway.

In Legionairre’s disease, the bacteria hijacks host _____ vesicles to look like _____, which allows for bacterial replication. This blocks delivery of _____ to ______.

Answer 19

EARLY ENDOSOME is the sorting system in the ENDOCYTIC pathway just as the TGN is the sorting system in the SECRETORY pathway.

In Legionairre’s disease, the bacteria hijacks host COPII vesicles to look like RER, which allows for bacterial replication. This blocks delivery of PHAGOSOME to LYSOSOME.

Question 20

Steps in receptor-mediated endocytosis:

1. ____ protein interacts with _____ receptor
2. Formation of ____ _____
3. Formation of ____ _____ via binding of ____ to tail of receptor on one side, and _____ on the other side.
4. Vesicle travels
5. Vesicle is ______
6. Vesicle is _____ and ready to ____

Answer 20

Steps in receptor-mediated endocytosis:

1. CARGO protein interacts with CARGO receptor
2. Formation of COATED PIT
3. Formation of COATED VESICLE via binding of ADAPTIN to tail of receptor on one side, and CLATHRIN on the other side.
4. Vesicle travels
5. Vesicle is UNCOATING
6. Vesicle is UNCOATED and ready to FUSE

Question 21

Following sorting in _____, receptors can be ____ or ____ in lysosome, while cargo can be delivered to _____ or _____ membrane via ______ to bypass _____ _____.

______ is used to deliver Abs to neonates.

Answer 21

Following sorting in EARLY ENDOSOME, receptors can be RECYCLED or DEGRADED in lysosome, while cargo can be delivered to LYSOSOME or BASOLATERAL membrane via TRANSCYTOSIS to bypass TIGHT JUNCTIONS.

TRANSCYTOSIS is used to deliver Abs to neonates.

Question 22

Examples of receptor and cargo trafficking following endocytosis:

1. ____ carries cholesterol through circulation as ____ complex.
2. The complex binds receptor, goes through endocytic pathway, fuses with endosome, and the ____ pH causes ___ dissociation from ____.
3. The ____ goes to lysosome, gets broken down into ____ _____ to be used by cell.
4. The ______ gets recycled and returned to the ______.

In contrast, EGF receptor gets ____ in order to ____-regulate the response. Example of _____ signaling.

Answer 22

Examples of receptor and cargo trafficking following endocytosis:

1. LDL carries cholesterol through circulation as LIPOPROTEIN complex.
2. The complex binds receptor, goes through endocytic pathway, fuses with endosome, and the LOW pH causes CARGO dissociation from RECEPTOR.
3. The LDL goes to lysosome, gets broken down into FREE CHOLESTEROL to be used by cell.
4. The RECEPTOR gets recycled and returned to the MEMBRANE.

In contrast, EGF receptor gets DEGRADED in order to DOWN-regulate the response. Example of LIMITED signaling.

Question 23

Mutation in LDL receptor ____ can prevent clearance because it will be unable to bind ______.

Answer 23

Mutation in LDL receptor TAIL can prevent clearance because it will be unable to bind ADAPTIN.

Question 24

Enveloped viruses like _____ utilize endocytic and biosynthetic pathways for propagation. It’s envelope proteins enter via _____-mediated endocytosis, the envelope fuses at ______ to release ____.

-Capsid proteins are made on ____ ribosomes and envelope proteins are made on ____ ribosomes (has _______).

Answer 24

Enveloped viruses like INFLUENZA utilize endocytic and biosynthetic pathways for propagation. It’s envelope proteins enter via CLATHRIN-mediated endocytosis, the envelope fuses at LOW PH to release CAPSID.

-Capsid proteins are made on FREE ribosomes and envelope proteins are made on BOUND ribosomes (has SRP RECOGNITION SEQUENCE).