Lecture 4

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

-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

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

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

mutation by making defective cells with lacking a specific glycosidase

would allow blocking of specif oligosacc modification

-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

-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

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

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

SNAP Receptors -bind to membrane component where NSF associates

Q is membrane 3 is needed R is vesicle just 1

Stalk hypothesis:

SNARE mediated SNARE-dependent fusion

-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)

-Coiled-coil tether -multi-subunit tether