The Newly Identified Yeast GRD Genes Are Required for Retention of Late-Golgi Membrane Proteins

Abstract

Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 complementation groups, were identified on the basis of their mislocalization of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the kinetics of A-ALP processing and by analyzing its localization by indirect immunofluorescence microscopy. The mutants were evaluated in a variety of other phenotypic tests relevant to yeast Golgi function, including processing of the ␣-factor mating pheromone, sorting of the vacuolar hydrolase carboxypeptidase Y, and retention of an early-Golgi membrane protein.Mutants from three grd complementation groups also failed to retain an early-Golgi membrane protein, suggesting that these mutations may have more global effects on Golgi retention and function. However, the majority of the grd mutants appeared to be defective specifically for the retention of several late-Golgi membrane proteins. A subset of the grd mutants appeared defective only in retention of A-ALP and not other late-Golgi membrane proteins. The grd mutants define a new set of genes required for Golgi membrane protein retention in S. cerevisiae.

ATPase-defective Mammalian VPS4 Localizes to Aberrant Endosomes and Impairs Cholesterol Trafficking

“…The ability of mutant hVPS4-transfected cells to sort both TGN38 and TfR from the early endosome appears at first perhaps more surprising, given that Vps4p-deficient yeast cells show significant defects in retention of late Golgi markers (Nothwehr et al, 1996). However, it is likely that yeast Golgi markers recycle via a later endocytic compartment than do mammalian TfR and TGN38.…”

“…Maturation of CPY to its vacuolar form is delayed and incomplete, and some CPY is missorted to the secretory pathway. Many class E vps mutants were also isolated in a genetic screen, which selected for mutants defective in efficient retention of late-Golgi proteins, which normally in yeast requires recycling of material from an endocytic compartment (Nothwehr et al, 1996). It therefore appears that sorting and/or transport of proteins from the class E compartment to both the vacuole and Golgi complex is defective in class E vps mutants.…”

“…The recent molecular characterization of one class E vps mutant, vps4, (Babst et al, 1997, also identified as end13, grd13, and csc1 (Munn and Riezman, 1994;Nothwehr The amino acid sequence of human VPS4 was compared with those of human and mouse SKD1 and yeast Vps4p. Identical amino acids are outlined in black.…”

Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast

“…The assay method performed on permeabilised whole cells using a-naphthyl phosphate (Sigma) as substrate was as previously described. 41 Fluorescence intensity was measured at 472 nm after excitation at 345 nm. Protein concentration was determined using BCA (Pierce).…”

Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

“…Parent strains (LCY81 and LCY82) in which a deletion of the END4/ SLA2 gene was covered by END4 on a URA3-based plasmid (pSLA2) were subjected to UV mutagenesis as described previously (Nothwehr et al, 1996) (Tables 1 and 2). Additional mutants were obtained by transforming the same strains with a NotI-digested yeast genomic library containing random insertions of a Tn3-LacZ transposon cassette (Burns et al, 1994).…”