Rainer Duden

The Sec34/35 complex was identified as one of the evolutionarily conserved protein complexes that regulates a cis-Golgi step in intracellular vesicular transport. We have identified three new proteins that associate with Sec35p and Sec34p in yeast cytosol. Mutations in these Sec34/35 complex subunits result in defects in basic Golgi functions, including glycosylation of secretory proteins, protein sorting, and retention of Golgi resident proteins. Furthermore, the Sec34/35 complex interacts genetically and physically with the Rab protein Ypt1p, intra-Golgi SNARE molecules, as well as with Golgi vesicle coat complex COPI. We propose that the Sec34/35 protein complex acts as a tether that connects cis-Golgi membranes and COPI-coated, retrogradely targeted intra-Golgi vesicles.

We examined the distribution of the non-clathrin-coated vesicle-associated coat protein beta-COP in rat exocrine pancreatic cells by immunogold cytochemistry. Labeling for beta-COP was found in the Golgi region (48%) where it was associated with vesicles and buds of approximately 50 nm, showing a characteristic approximately 10-nm-thick coat. The other half of the label was present in the cytoplasm, not associated with visible coats or membranes, with a minor fraction present on small clusters of tubules and vesicles. Clathrin-coated vesicles were typically located at the trans-side of the Golgi complex, and showed a thicker coat of approximately 18 nm. Of the total beta-COP labeling over the Golgi region, 68% occurred on the cis-side, 6% on the cisternae, 17% on the rims of the cisternae, and only 9% on the trans-side. For clathrin these figures were 16, 2, 4, and 78%, respectively. At the cis-Golgi side beta-COP was present in transitional areas (TA), on so-called peripheral elements (PE), consisting of tubules and vesicles located between the cup-shaped transitional elements (TE) of the RER and the cis-most Golgi cisternae. Label for Sec23p was also present in TA but was located closer to the TE, while beta-COP labeled PE were located near the cis-Golgi cisternae. Upon energy depletion, Golgi associated beta-COP was almost exclusively (86%) in spherical aggregates of 200-500 nm in diameter, whereas the cis-side (6%), the cisternae (1%), the rims (4%) and trans-side (3%) of the Golgi complex, were barely labeled; 50% of the total label remained in the cytoplasm. The aggregates were predominantly located at the cis-side of the Golgi stack, next to, but distinct from the Sec23p positive TA, that were devoid of beta-COP and had only a few recognizable vesicles left. Incubation with aluminum fluoride resulted in fragmentation of the Golgi complex into large clusters of beta-COP positive vesicles, while 50% of the label remained in the cytoplasm, as in control cells. After 10 min of Brefeldin A treatment 91% of beta-COP was cytoplasmic and only 7% associated with membranes of the Golgi complex. The total label for beta-COP over exocrine cells remained unchanged during the incubation with either of the drugs, indicating that the drugs induce reallocation of beta-COP. Our data suggest that beta-COP plays a role in membrane transport at the cis-side of the Golgi complex.