Research Summary

Our main goal is to understand the processes that generate Golgi stacks. The cisternal maturation model provides a conceptual framework for studying Golgi formation. This model postulates that new Golgi elements arise at transitional ER (tER) sites, which are specialized for the production of ER-to-Golgi transport vesicles. We have obtained evidence that in budding yeasts, Golgi distribution is a consequence of tER organization. In Saccharomyces cerevisiae, Golgi cisternae are dispersed throughout the cytoplasm and the entire ER network functions as tER, whereas in Pichia pastoris, ordered Golgi stacks are located next to discrete tER sites. We are analyzing these two yeasts in parallel with vertebrate cells. Our specific approaches are: (1) To characterize the inheritance and dynamics of Golgi cisternae in S. cerevisiae through a combination of genetics and 4D video microscopy. (2) To study tER organization and biogenesis in P. pastoris using genetics, molecular biology, video microscopy, and biophysical computer simulations. P. pastoris is an ideal model organism for these studies. (3) To explore tER organization and dynamics in vertebrate cells. This approach is revealing evolutionarily conserved mechanisms that generate tER sites.

A second project in the lab involves optimizing the red fluorescent protein DsRed. Like GFP, DsRed potentially has wide application as a reporter and fusion tag. However, wild-type DsRed matures very slowly, requiring more than 24 hours at 37 C to achieve maximal fluorescence. We overcame this problem by using directed evolution to create rapidly maturing DsRed variants, one of which is now marketed commercially as DsRed-Express. Wild-type DsRed also tetramerizes, limiting its usefulness as a fusion tag. Ongoing work is aimed at creating a monomeric DsRed variant that will be as versatile as GFP.

Selected Publications

Losev E, Reinke CA, Jellen J, Strongin DE, Bevis BJ, Glick BS. (2006) Golgi maturation visualized in living yeast. Nature 441: 1002-1006. (PubMed)

Connerly PL, Esaki M, Montegna EA, Strongin DE, Levi S, Soderholm J, Glick BS. (2005) Sec16 is a determinant of transitional ER organization. Curr. Biol. 15: 1439-1447. (PubMed)

Soderholm J, Bhattacharyya D, Strongin D, Markovitz V, Connerly PL, Reinke CA, Glick BS. (2004). The transitional ER localization mechanism of Pichia pastoris Sec12. Dev Cell 6: 649-659. (PubMed)

Bonifacino J, Glick BS. (2004). The mechanisms of vesicle budding and fusion. Cell 116: 153-166. (PubMed)

Bevis BJ, Hammond AT, Reinke CA, Glick BS. (2002). De novo formation of transitional ER sites and Golgi structures in Pichia pastoris. Nat Cell Biol 4: 750-756. (PubMed)

Bevis BJ, Glick BS. (2002). Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat Biotechnol 20: 83-87. (PubMed)

Hammond AT, Glick BS. (2000). Dynamics of transitional endoplasmic reticulum sites in vertebrate cells. Mol Biol Cell 11: 3013-30. (PubMed)

Rossanese OW, Soderholm J, Bevis BJ, Sears IB, O'Connor J, Williamson EK, Glick BS. (1999). Golgi structure correlates with transitional endoplasmic reticulum organization in Pichia pastoris and Saccharomyces cerevisiae. J Cell Biol 145: 69-81. (PubMed)