“Reappraisal of the role of nucleoside diphosphatases on yeast N- and O-glycosylation”

CECILIA D'ALESSIO; ARMANDO J. PARODI

Mérida, México

Congreso; 2005 Meeting of International Resarch Scholars of the Howard Hughes Medical Institute.; 2005

Howard Hughes Medical Institute

Nucleoside diphosphatases (NDPases) are required to relieve glycosyltransferase inhibition by nucleoside diphosphates (NDPs) and to provide nucleoside monophosphates that function as antiporters in nucleotide sugar entrance into the secretory pathway. We previously communicated that in Schizosaccharomyces pombe, a yeast with an active UDP-Glc:glycoprotein glucosyltransferase (GT) in the endoplasmic reticulum (ER), both secretory pathway NDPases localized to the Golgi. To study how yeast cells dispose of NDPs generated in the ER, we have now switched to Saccharomyces cerevisiae, a yeast that displays only two Golgi NDPases in the secretory pathway (Gda1p and Ynd1p). We found that S. pombe GT expressed in S. cerevisiae ER was functional in vivo in mutants lacking both NDPases and at the restrictive temperature in conditional mutants affected in ER-Golgi vesicular transport. Moreover, it was found that Gda1p_/Ynd1p_ double mutants were able to add a single Man unit to Ser/Thr-Man and two to three Man residues to Asn-GlcNAc2Man8. By contrast, single mutants formed both O- and N-linked glycans displaying normal, wild-type cell sizes. Labeling conditional mutant cells at the restrictive temperature showed that the first nucleotide sugar-dependent addition of a Man unit in O-glycosylation occurred in the ER, whereas addition of the first two to three Man units to Asn­GlcNAc2Man8 took place in the cis-Golgi. It was concluded that (1) UDP-Glc and GDP-Man enter the ER lumen in the absence of NDPases in the secretory pathway and of anterograde and retrograde vesicular transport; (2) formation of Ser/ Thr-Man2 occurs in the ER; (3) the first steps of nucleotide sugar-dependent N-glycan elongation occur in the cis-Golgi, even in the absence of NDPases; (4) GDP-Man enters the cis-Golgi in the absence of both Gda1p and Ynd1p; and (5) NDPases are strictly required for further addition of Man units to Ser/Thr-Man2 and Asn-GlcNAc2Man10-11, processes that presumably take place in the medial and trans-Golgi.