LEcture 8

Sorts proteins (lipids) destined for TGN (main site of sorting) Modifies N- and O-linked oligos (Carbohydrate factory)

has beautiful Golgi

They stain differently because their biochemically compartmentalized

Phosphorylation of lysosomal enzumes

Removal of Mannose Addition of GlcNAc

Addition of Galactose and Sialic Acid

Type I: has N-terminus at lumen found in TGN Type II: has N-terminus at cytosol intra-golgi and are Glycosylation enzymes

Hypothesis is Kin recognition. They associate with their own proteins and form higher-oligomer structure. cis and cis This is not possible as they can’t enter vesicle due to size constraints

Fluorescene Loss in Photobleaching -used to study connectivity

Fluorescence Gain After Photobleaching -used to study diffusion (time)

Using FLIP, they found Golgi enzymes diffusing freely and rapidly with intra-Golgi network

Uses immunofluorescence to image tagged proteins using antibodies

unlabeled primary antibody allows multiple secondary (use different species for antigen)

Fixing is to maintain cell structure and antigenicity Formaldehyde is reversible but weak Methanol is organic and denatures proteins Acetone is organic and preserves proteins

Pinhole allows pixel by pixel good quality

cis medial trans TGN ERGIC

ER-Golgi Intermediate Compartment at 15C it accumulates secreted proteins

They guide ERGIC to Golgi from transitional (tER)

Golgi160 is the tether for dynein and ERGICis microtubule-dependent motor

COPII exits from ER to ERGIC COPI present in ERGIC and brings back to ER

BFA inhibits exchange factor GEF of Arf1 which cannot recruit COPI to Golgi Golgi fuses with ER and when BFA is washed Golgi reappears

Standard Model Maturation Model Intra-Golgi Recycling