Yeast Functional Analysis: Identification of Two Essential Genes Involved in ER to Golgi Trafficking

Abstract

We screened for genes potentially involved in the secretory and vacuolar pathways a collection of 61 yeast strains, each bearing an essential orphan gene regulated by the tetO 7 -CYC1 promoter that can be down-regulated by doxycycline. After down-regulating the expression of these genes, we performed systematic Western blot analysis for markers of the secretory and vacuolar pathways that undergo post-translational modifications in their intracellular trafficking. Accumulation of protein precursors, revealed by Western immunoblot analysis, indicates defects in the secretory pathway or in associated biochemical modifications. After screening the whole collection, we identified two genes involved in ER to Golgi trafficking: RER2, a cis-prenyl transferase, and USE1, the function of which was unknown. We demonstrated that repression of USE1 also leads to BiP secretion, and therefore likely affects retrograde, in addition to anterograde, ER to Golgi trafficking. The collection also includes two essential genes involved in intracellular trafficking that were conveniently repressed without resulting growth or trafficking defects.

Effect of salt stress on prenol lipids in the leaves of Tilia 'Euchlora'

“…The latter process has been studied in rat liver (Thelin et al 1995), spinach (Swiezewska et al 1993 and Arabidopsis thaliana (Gutkowska et al 2004). Genetic studies on yeast have shown that polyisoprenoids are involved in protein trafficking to the endoplasmic reticulum (ER) and vacuole (Sato et al 1999;Belgareh-Touze et al 2003). The role of free polyisoprenoid alcohols and their carboxylic esters remains uncertain.…”

Dsl1p, Tip20p, and the Novel Dsl3(Sec39) Protein Are Required for the Stability of the Q/t-SNARE Complex at the Endoplasmic Reticulum in Yeast

“…The zippering of the four membrane proximal SNARE motifs results in close apposition of the vesicle and target membranes, perhaps providing the means by which SNAREs directly mediate membrane fusion in vitro. SNAREs implicated in the fusion of Golgi derived vesicles with the ER membrane are Ufe1p, Use1p, Sec20p and Sec22p (Sweet and Pelham, 1992;Lewis and Pelham, 1996;Cosson et al, 1997;Belgareh-Touze et al, 2003;Burri et al, 2003;Dilcher et al, 2003). During trans-SNARE pairing, these four SNARE proteins would form a four helix bundle whose central polar layer is made up by one arginine residue derived from Sec22p, two glutamine residues (Ufe1p, Sec20p) and one aspartate (Use1p).…”

What We Do and Do Not Know About the Cellular Functions of Polyisoprenoids

“…The involvement of polyisoprenoids in intracellular traf fi c of proteins was suggested (Sato et al 1999 ) . Later, dolichols were considered as being essential for anterograde vesicle traf fi cking (Belgareh-Touze et al 2003 ) . Very recently, an impairment of dolichol phosphorylation has been recognized as a cause of a new inherited human disorder, and severely diminished protein glycosylation was considered as a major reason of metabolic dysfunctions, whereas protein prenylation had not been analyzed (Kranz et al 2007 ) .…”