Topography of glycosylation in yeast: characterization of GDPmannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles.

Abstract

ABSTRACT"Outer-chain" addition of mannose sidues to yeast glycoproteins occurs in the Golgi compartment of the cell. Essential steps in this process are thought to indude transport of GDPmannose from the cytoplasm into the lumen of Golgi vesicles, transfer of mannose to glycoprotein acceptors, hydrolysis of the resulting GDP to GMP, and return of GMP and inorganic phosphate to the cytoplasm. We report detection and characterization of a GDPmannose transport activity and a GDPase by yeast vesicles. The active transport of GDPmannose as well as the GDPase and another presumed Golgi enzyme, al,2-mannosyliransferase, are concentrated in a subcellular fraction that can be partially separated, by velocity sucrose gradient centrifugation, from a fraction enriched in an endoplasmic reticulum marker enzyme. Subcellular Fractionation. The dolichyl-phosphatemannose (Dol-P-Man) synthase mutant XD2-7C (10), grown at 300C was used in all experiments except those where a velocity sucrose gradient fractionation was done (see Fig. 3)..Cells were grown in YPD medium to an OD6w of 4. The 800-ml culture was chilled, centrifuged, and converted to spheroplasts, as described by Walworth and Novick (11), with a total of 10 mg of Zymolyase 100T (ICN). The spheroplast suspension was then centrifuged at 450 x g for 10 min. The cells were broken by suspending each pellet in 10 ml of 10 mM TEA, pH 7.2/0.8 M sorbitol/1 mM EDTA. The Abbreviations: NDPase, nucleoside diphosphatase; Dol-P-Man, dolichyl-phosphate mannose; TEA, triethanolamine acetate. 6935The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast

“…which had been reported previously by others as characteristic for these organelles (Abeijon et al, 1989;Sanderson and Meyer, 1991 ;Antebi and Fink, 1992). The vacuolar marker enzyme a-mannosidase is clearly separated from the marker enzymes of the secretory pathway (Fig.…”

“…Subcellular fractionation was performed as described by Abeijon et al (1989), modified by Antebi and Fink (1992). In a modification to Antebi and Fink the spheroplasts were formed with Zymolyase 100-T and only N-tosyl-~-phenylalanylchloromethane and phenylmethylsulfonyl fluoride were used as inhibitors.…”

Nucleoside Diphosphatase and Glycosyltransferase Activities Can Localize to Different Subcellular Compartments in Schizosaccharomyces pombe

“…Nucleoside phosphatase activities were essentially assayed as described with slight modifications (21). Briefly, between 10 and 50 g of membrane proteins were incubated in a total volume of 100 l in 0.2 M imidazole buffer, pH 7.2, 0.1% digitonin, 10 mM CaCl 2 , 2 mM the corresponding nucleoside phosphate, and incubated for 5-10 min at 30°C.…”

QSR1, an Essential Yeast Gene with a Genetic Relationship to a Subunit of the Mitochondrial Cytochromebc 1 Complex, Codes for a 60 S Ribosomal Subunit Protein

“…The GDPase marker for the Golgi apparatus was assayed enzymatically (19), and the maximum activity was scaled to 100%. Marker recoveries were calculated as activity per l ϫ volume divided by activity per l of lysate ϫ volume of lysate for each marker.…”