Saccharomyces cerevisiae has been the organism of choice for genetic studies of secretion, but S. cerevisiae Golgi shows an unusual organization: rather than forming ordered stacks, the cisternae are scattered throughout the cytoplasm. By contrast, the related budding yeast Pichia pastoris has conventional Golgi stacks. Dr. Glick and his lab have proposed that P. pastoris contains discrete tER sites that repeatedly generate Golgi cisternae, and form stacks. On the other hand, S. cerevisiae lacks tER sites; the entire ER network functions as tER, suggesting COPII vesicles bud throughout the ER and generate Golgi cisternae throughout the cytoplasm. Their initial tests of this hypothesis have confirmed that Golgi organization is fundamentally different in the two yeasts, and that P. pastoris contains discrete tER sites whereas S. cerevisiae does not. Thus, P. pastoris is an ideal model organism for studying the mechanisms that generate discrete tER sites and ordered Golgi stacks.

Dr. Glick’s current efforts are focused on characterizing the formation and dynamics of tER sites in P. pastoris. For this purpose he has rendered tER sites fluorescent by fusing GFP to the Sec13 subunit of the COPII coat. To identify genes responsible for organizing tER sites, he has used the P. pastoris strain carrying Sec13-GFP to isolate mutants with altered tER organization. In parallel, Dr. Glick is monitoring tER dynamics by rapid three-dimensional time-lapse (4D) confocal microscopy. His 4D movies have revealed that tER sites are long-lived structures that form de novo, grow to full size, and occasionally fuse. These data suggest that tER sites are dynamic protein-lipid “patches” that assemble and diffuse in the plane of the ER membrane.

Many of the phenomena that Dr. Glick sees with P. pastoris appear to be conserved in evolution. For example, mammalian cells also contain discrete tER sites, although a typical mammalian cell has about 100 times as many tER sites as a P. pastoris cell. These basic studies should yield important clues to trafficking in mammalian cells such as the pancreatic β-cell.