Three-dimensional and immune electron microscopic analysis of the secretory pathway in Saccharomyces cerevisiae

Until now, the mechanisms of ER-to-Golgi and intra-Golgi transport remain obscure. This is especially evident for the Golgi of S. cerevisiae where different Golgi compartments are not organized in stacks. Here, using improved sample preparation protocols, we examined the 3D organization of pre-Golgi and Golgi compartments and found several new features of the structures functioning along the secretory pathway. In the cytoplasmic sheet ER, we found narrow pores that aggregated near the rims, and tubular networks tightly interconnected with sheets of several cytoplasmic ER cisternae. Within the Golgi compartments, we found disks with wide pores, disks with narrow pores, and disk-like networks with varicosities or nodules at the point of branching and thick membranes. Sometimes, these compartments contained 30 nm buds coated with a clathrin-like coat. The lumen of these Golgi compartments was more osmiophilic than the lumen of the ER. In contrast to ER elements, Golgi compartments were isolated and in the majority of cases not connected, although we observed some connections between Golgi compartments and also between Golgi disks with wide pores and the ER. Two types of free vesicles of 35-40 and 45-50 nm were found, the former being sometimes partially coated with a clathrin-like coat. Sec31, a COPII component, was found near narrow pores in the cytoplasmic sheets of the ER, over edge aggregates of narrow pores, and within the ER network. The cis-Golgi marker Rer1p was detected on disks or semi-spheres with wide pores, while the medial Golgi marker Gos1p was found on disks or semi-spheres with narrow pores. Gos1p was found to be enriched on 45-50 nm vesicles, while Rer1p was depleted. The 35-40 nm vesicles did not show either label. These findings are discussed from the point of view of mechanisms of transport.