Lecture 4

Question 1

Glycosylation

Answer 1

Oligosaccharide formation occurs in the Golgi
-attaching of carbohydrates to proteins and lipids
-N-linked glycosylation Asn-X-Ser/Thr

Question 2

Tools for studying glycosylation

Answer 2

-identifying glycosylation state of proteins indicate enzymes they’ve encountered, these enzymes are compartmentalized
-identifying their topology by introducing or removing glycosylation sites
-lectins bind specific sugars

Question 3

O-linked vs N-linked

Answer 3

N-linked (Asparagine) are in HP
O-linked (Serine/Threonine) cytosolic

Question 4

Glycosidases

Answer 4

enzymes specific for N- and O-linked glycans
- treatments leads to molecular shift on SDS-PAGE
sensitivity to enzyme indicates its compartment
-EndoH cleaves high mannose (early oligos) a molecular shift would mean it is still around cis and not reached medial cisterna

Question 5

CHO cells

Answer 5

mutation by making defective cells with lacking a specific glycosidase

Question 6

Inhibitors

Answer 6

would allow blocking of specif oligosacc modification

Question 7

Rothman Assay

Answer 7

-helped us understand Intra-Golgi Transport
Original: involved 15B mutant donor cells with VSV-G treated with radioactive label and has no GlcNAc transferase with an acceptor cell that was wild-type with the GlcNAc tranferase. If the radioactive label was transfered on to the acceptor cell with a glycosylated GlcNAc that confirms glycosylation

Question 8

Updated Assay with NEM

Answer 8

-Found NSF or NEM-sensitive-factor that acts as transport component
-Desalting, KCl led to destabilization of ATP which is need for stabilization of NSF

Question 9

NSF

Answer 9

-NEM-sensitive-factor
-AAA protein fam: ATPases Associated with diverse cellular activities
-Helps with remodeling protein complexes

Question 10

SNAP

Answer 10

Soluble NSF Attachment Protein
-promotes NSF binding
-identified by washing Golgi with high salt and breaking electrostatic interactions with proteins, attachment was SNAP

Question 11

SNAREs

Answer 11

SNAP Receptors
-bind to membrane component where NSF associates

Question 12

Q- and R- SNAREs

Answer 12

Q is membrane 3 is needed
R is vesicle just 1

Question 13

SNARE-mediated fusion

Answer 13

Stalk hypothesis:

Question 14

SM Proteins

Answer 14

SNARE mediated
SNARE-dependent fusion

Question 15

Rab GTPase cycle

Answer 15

-Brought by GDI to membrane
-Activated by GEF Rab-GTP
-Rab then attaches with effectors like…
-Rab is recycled back and hydrolyzed by GAP (GTPase activating protein)

Question 16

Vesicle Tethers

Answer 16

-Coiled-coil tether
-multi-subunit tether