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 ER contains multiple small tER sites, whereas in Pichia pastoris, ordered Golgi stacks are located next to large, stable tER sites. We are analyzing these two yeasts in parallel with vertebrate cells. Our specific approaches are: (1) To characterize the 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, and video microscopy. 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. More recent work yielded a noncytotoxic variant called DsRed-Express2, as well as a far-red variant called E2-Crimson. In parallel with this protein engineering work, we are collaborating with Bob Keenan's group to study basic mechanisms of chromophore formation in DsRed and other fluorescent proteins.
Strack RL, Strongin DE, Mets L, Glick BS, Keenan RJ. (2010) Chromophore formation in DsRed occurs by a branched pathway. J Am Chem Soc 132:8496-8505. (PubMed)
Strack RL, Hein B, Bhattacharyya D, Hell SW, Keenan RJ, Glick BS. (2009) A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling. Biochemistry 48:8279-8281. (PubMed)
Glick BS, Nakano A. (2009) Membrane traffic within the Golgi apparatus. Annu Rev Cell Dev Biol. 25: 113-132. (PubMed)
Strack RL, Strongin DE, Bhattacharyya D, Tao W, Berman A, Broxmeyer HE, Keenan RJ, Glick BS. (2008) A noncytotoxic DsRed variant for whole-cell labeling. Nat Methods 5:955-957. (PubMed)
Bhattacharyya D, Glick BS. (2007) Two mammalian Sec16 homologues have nonredundant functions in endoplasmic reticulum (ER) export and transitional ER organization. Mol Biol Cell 18:839-849. (PubMed)
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
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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)
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of transitional endoplasmic reticulum sites in vertebrate cells.
Mol Biol Cell 11: 3013-30. (PubMed)
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