Endomembranes I: Moving Proteins and Membranes in the Secretory Pathway

Question 1

Principles of vesicle trafficking

Answer 1

1) Cargo selection
2) Vesicle budding from one compartment
3) Transport
4) Fusion with a second compartment

Question 2

Studies that used ___ established understanding of the molecular mechanisms involved in vesicle trafficking.

Answer 2

biochemical, subcellular fractionation, and genetic approaches

Question 3

Protein transport through the secretory pathway can be visualized by ____.

Answer 3

fluorescence microscopy of cells producing a GFP-membrane fusion protein (fluorescent molecule movement tracked over time)

Question 4

Transport of a membrane glycoprotein (VSVG) can be assayed by ____.

Answer 4

Sensitivity to endoglycosidase D

Question 5

Transport of ___ can be assayed by sensitivity to endoglycosidase D.

Answer 5

a membrane glycoprotein (VSVG)

Question 6

Yeast Sec mutant mutation experiment lead to what discovery?

How did it work?

Answer 6

Tracking of the secreted protein invertase in S. cerevisiae, and identification of ts mutants with defects at various steps, led to the discovery of numerous players in vesicle trafficking.
Conditional or temperature-sensitive (ts) mutants secrete invertase at permissive, lower temperature but accumulate proteins in the cell at restrictive or high temp.

Question 7

Yeast sec mutants

Class, defect, and fate of protein

Answer 7

Class A:
Fate of proteins (FoP): Accumulation in the cytosol, Defect (D): transport into the ER
Class B:
FoP: Accumulation in rough ER, D: Budding of vesicles from the rough ER
Class C:
FoP: Accumulation in ER-to-Golgi transport vesicles, D: Fusion of transport vesicles with golgi
Class D:
FoP: Accumulation in Golgi, D: Transport from Golgi to secretory vesicles
Class E:
FoP: Accumulation in secretory vesicles, D: Transport from secretory vesicles to cell surface

Question 8

In normal mammalian cells, VSVG is modified in the Golgi by ____.

Answer 8

an GlcNAc transferase

Question 9

In normal mammalian cells, ____ is modified in the Golgi by GlcNAc transferase.

Answer 9

VSVG

Question 10

How does a cell-free assay work?

Answer 10

Isolated Golgi stacks from uninfected wild-type and infected mutant cell lines are combined in the test tube, then VSVG is glycosylated.
It showed intercompartmental transport.
Assumes that vesicles transport cargo (VSVG) or enzymes between golgi.

From what I understand, when the mutant cell was incubated with the wild type cell, the N-Acetylglucosamine was added to the protein of interest. When the cells were separate, N-acetylglucosamine was not added to the protein of interest.

Question 11

The addition of N-acetylglucosamine requires ___.

Answer 11

Transport vesicles, cytosol, and ATP and GTP

Question 12

Mechanical forces used in vesicle transport:

Answer 12

Scission, propulsion/motility

Question 13

Target recognition protein

Answer 13

Rab GTPases

Question 14

Membrane function protein

Answer 14

SNAREs

Question 15

____ drives vesicle budding and selection of cargo.

Answer 15

Coat assembly

Question 16

Cargo can be ____ or ___.

Answer 16

soluble, luminal proteins or integral membrane proteins

Question 17

Vesicle Coat types:
Transport Step Mediated
Coat proteins
Associated GTPase

Answer 17

COPII:
ER to cis-Golgi; Sec23/Sec24 and Sec13/Sec31 complexes, Sec16; Ser1
COPI:
cis-Golgi to ER, later to earlier Golgi cisternae; Coatamers containing seven different COP subunits; ARF
Clathrin and adapter proteins:
trans-Golgi to endosome; Clathrin and AP1 complexes or Clathrin and GGA; ARF
Plasma membrane to endosome; Clathrin and AP2 complexes; ARF
Golgi to lysosome, melanosome, or platelet vesicles; AP3 Complexes; ARF

Question 18

COPII:
Transport Step Mediated
Coat proteins
Associated GTPase

Answer 18

ER to cis-Golgi;
Coat Proteins: Sec23/Sec24 and Sec13/Sec31 complexes, Sec16;
Associated GTPase: Ser1

Question 19

COPI:
Transport Step Mediated
Coat proteins
Associated GTPase

Answer 19

cis-Golgi to ER, later to earlier Golgi cisternae;
Coat proteins: Coatamers containing seven different COP subunits;
Associated GTPase: ARF

Question 20

Clathrin and adapter proteins:
Transport Step Mediated
Coat proteins
Associated GTPase

Answer 20

trans-Golgi to endosome; Clathrin and AP1 complexes or Clathrin and GGA; ARF

Plasma membrane to endosome; Clathrin and AP2 complexes; ARF

Golgi to lysosome, melanosome, or platelet vesicles; AP3 Complexes; ARF

Question 21

SAR1 and ARF are members of ____.

Answer 21

the Ras GTPase superfamily

Question 22

What proteins affect GTPase?

Answer 22

GEF activates GTPases, GAP deactivates GTPases

Question 23

Cytoplasmic Sar1 is activated and recruited to ER membrane by ____.

Answer 23

Sec12, an integral membrane protein

Question 24

Sec12 is a ___ for ___; catalyzes the exchange of ___ for ___.

Answer 24

GEF for Sar1

GDP for GTP

Question 25

GTP-Sar1 undergoes a conformational change, exposing the ____ for membrane association. ARF has an ___.

Answer 25

hydrophobic N-terminus, | N-terminal myristate group

Question 26

GTP-Sar1 recruits _____ to the membrane.

Answer 26

the heterodimeric Sec23/Sec24 coat (COPII) proteins

Question 27

___ recruits cargo to the vesicle budding site

Answer 27

Sec24

Question 28

____ joins the coat and self-polymerizes into lattices

Answer 28

Sec13 and Sec31 heterodimer

Question 29

Sec23 is a ___ for ___; stimulates ____ after coat assembly

Answer 29

GAP for Sar1, | GTP hydrolysis

Question 30

COPI Signal Sequence Proteins with Signal Signal Receptor

Answer 30

Lys-Asp-Glu-Leu (KDEL); ER-resident soluble proteins; KDEL receptor in cis-golgi membrane Lys-Lys-X-X; ER-resident soluble proteins; COPI alpha and beta subunits

Question 31

COPII Signal Sequence Proteins with Signal Signal Receptor

Answer 31

Di-acidic (e.g. Asp-X-Glu); Cargo membrane proteins in ER; COPII Sec24 subunit

Question 32

Clathrin/AP1 Signal Sequence Proteins with Signal Signal Receptor

Answer 32

Mannose-6-phosphate; soluble lysosomal enzymes after processing in cis-Golgi; M6P receptor in trans-golgi membrane Tyr-X-X-Sai; Membrane proteins in trans-Golgi; AP1 (mew1 subunit)

Question 33

Clathrin/AP2 Signal Sequence Proteins with Signal Signal Receptor

Answer 33

Mannose-6-phosphate; secreted lysosomal enzymes; M6P receptor in plasma membrane Asn-Pro-X-Tyr (NPXY); LDL receptor in plasma membrane; AP2 complex Tyr-X-X-Sai; Plasma membrane proteins; AP2 (mew2 subunit) Leu-Leu; Plasma membrane proteins; AP2 complexes

Question 34

Mannose-6-Phosphate Proteins with signal Signal Receptor Vesicles that incorporate Signal-bearing protein

Answer 34

soluble lysosomal enzymes after processing in cis-Golgi; M6P receptor in trans-golgi membrane; Clathrin/AP1 secreted lysosomal enzymes; M6P receptor in plasma membrane; Clathrin/AP2

Question 35

Lys-Asp-Glu-Leu Proteins with signal Signal Receptor Vesicles that incorporate Signal-bearing protein

Answer 35

ER-resident soluble proteins; KDEL receptor in cis-golgi membrane; COPI

Question 36

Di-acidic (e.g. Asp-X-Glu) Proteins with signal Signal Receptor Vesicles that incorporate Signal-bearing protein

Answer 36

Cargo membrane proteins in ER; COPII Sec24 subunit; COPII

Question 37

Cystic Fibrosis

Answer 37

CFTR, encodes an integral membrane protein that is normally transported to the PM through the secretory pathway in epithelial cells CFTR is a chloride channel that has di-acidic sorting signal for packaging in COPII vesicles at the ER. A common mutation, ΔF508, blocks export of CFTR from the ER, perhaps by changing the conformation or exposure of the di-acidic sorting signal so that it can no longer bind to Sec24.

Question 38

Like Sar/Arf, GTP-Rab undergoes a conformational change to expose an ___ that associates with membranes

Answer 38

prenylated anchor

Question 39

Steps of fusion of transport vesicle with cell membrane

Answer 39

Step 1 : A Rab protein tethered via a lipid anchor to a secretory vesicle binds to an effector protein complex on the plasma membrane, thereby docking the transport vesicle on the appropriate target membrane. Step 2 : A v-SNARE protein (in this case, VAMP} interacts with the cytosolic domains of the cognate t-SNAREs (in this case, syntaxin and SNAP-25}. The very stable (oiled-coil SNARE complexes that are formed hold the vesicle close to the target membrane. Step 3: Fusion of the two membranes immediately follows formation of SNARE complexes, but precisely how this occurs is not known. Step 4: Following membrane fusion, NSF in conjunction with alpha-SNAP protein binds to the SNARE complexes. The NSF-catalyzed hydrolysis of ATP then drives dissociation of the SNARE complexes, freeing the SNARE proteins for another round of vesicle fusion. Also at this time, Rab-GTP is hydrolyzed to Rab-GDP and dissociates from the Rab effector (not shown}.

Question 40

Functions of the golgi apparatus

Answer 40

Transport and Packaging | Modification and Synthesis of oligosaccharides

Question 41

The ___ face of the golgi is the forming face and the ___ face is the maturing face.

Answer 41

cis, | trans

Question 42

``` Lys-Lys-X-X Proteins with signal Signal Receptor Vesicles that incorporate Signal-bearing protein Where contents go ```

Answer 42

ER-resident soluble proteins; COPI alpha and beta subunits; COPI; Golgi to ER

Question 43

______ of luminal proteins, like BiP or PDI, is necessary & sufficient to allow ER localization.

Answer 43

KDEL or HDEL at the C-terminus

Question 44

Mis-sorted ____ are recovered from cis-Golgi by a ___ receptor that interacts with COPI coats–retrograde transport.

Answer 44

KDEL proteins, | KDEL

Question 45

Cisternal maturation model of maturation/trafficking through the Golgi apparatus

Answer 45

Cisterna form on one side, mature and move through the stack. Cargo stay with its cisterna (no forward vesicle trafficking) Enzymes move backward by retrograde flow (COPI vesicles)

Question 46

Which is the correct model of maturation/trafficking through the Golgi apparatus?

Answer 46

``` Cisternal maturation (not Vesicle Shuttling) Evidence is Algal scales and collagen fibrils ```

Question 47

Clathrin is associated with protein transport to ___.

Answer 47

Late endosomes

Question 48

AP3 is associated with protein transport to ___.

Answer 48

lysosomes

Question 49

Enzyme activities required to modify one or more mannose residues on C-6, generating M6P

Answer 49

GlcNAc phosphotransferase, has a binding site for lysosomal protein recognition. A phosphodiesterase trims off GlcNAc.

Question 50

``` Tyr-X-X-Sai Proteins with signal Signal Receptor Vesicles that incorporate Signal-bearing protein Where contents go ```

Answer 50

Membrane proteins in trans-Golgi; AP1 (mew1 subunit); Clathrin/AP1; Contents moved to late endosome Plasma membrane proteins; AP2 (mew2 subunit); Clathrin/AP2; Contents moved to late endosome

Question 51

M6P Receptor unloads/ releases its cargo under what condition?

Answer 51

At acidic pH

Question 52

Lysosomal storage diseases

Answer 52

Tay Sachs, Mucopolysaccharide Storage and I-cell disease Caused by absence of one or more lysosomal enzymes. Thus, undigested glycolipids and extracellular components accumulate in lysosomes as large inclusions.

Question 53

I-Cell Disease

Answer 53

Caused by loss of multiple enzymes. Patients lack N-acetylgluosamine phosphotransferase required for formation of M6P on lysosomal enzymes in the cis-Golgi. This leads to secretion of the lysosomal enzymes and lack of proper lysosomal action. Patients suffer from abnormalities of face/skull; growth delays; mental retardation. Study of I-cell disease patients was valuable in determining the fact that M6P targeted enzymes to the lysosome and that such enzymes were sometimes secreted and then taken up and incorporated into lysosomes.