Identification of a human orthologue of Sec34p as a component of the cis-Golgi vesicle tethering machinery

The roles of the components of the Sec34p protein complex in intracellular membrane trafficking, first identified in the yeast Saccharomyces cerevisia e, have yet to be characterized in higher eukaryotes. We cloned a human cDN A whose predicted amino acid sequence showed 41% similarity to yeast Sec34p with homology throughout the entire coding region. Affinity-purified antib odies raised against the human SEC34 protein (hSec34p) recognized a cellula r protein of 94 kDa in both soluble and membrane fractions. Like yeast Sec3 4p, cytosolic hSec34p migrated with an apparent molecular mass of 300 kDa o n a glycerol velocity gradient, suggesting that it is part of a protein com plex. Immunofluorescence microscopy localized hSec34p to the Golgi compartm ent in cells of all species examined, where it co-localized well with the c is/medial Golgi marker membrin and partially co-localized with cis-Golgi ne twork marker p115 and trans-Golgi marker TGN38. The co localization with me mbrin was maintained at 15 degreesC and after microtubule depolymerization with nocodazole. During transport of the tsO45 vesicular stomatitis virus G protein through the Golgi, there was significant overlap with the hSec34p compartment. Green fluorescent protein-hSec34 expressed in HeLa cells was r estricted to Golgi cisternae, and its membrane association was sensitive to brefeldin A treatment. Taken together, our findings indicate that hSec34p is part of a peripheral membrane protein complex localized on cis/medial Go lgi cisternae where it may participate in tethering intra-Golgi transport v esicles.