Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligosaccharide content of the individual sequons.

Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates.

“…However, the relatively modest underglycosylation of invertase, which contains three N-X-S sites out of thirteen total sites, does not provide strong support for the hypothesis that Aost3 mutants are selectively deficient in glycosylation of N-X-S sites. A thorough analysis of the carbohydrate content of each of the glycosylation sequons in invertase has revealed that seven sites are modified in all invertase molecules synthesized by wild-type yeast, while five additional consensus sites are subject to variable glycosylation ranging between 30 and 80% (Reddy et al, 1988). We have not determined which sites in invertase are subject to underglycosylation by the Aost3 mutant, so it is not clear whether the reduction in glycosylation is a consequence of reduced modification at all normally used sites, or is instead due to selective underglycosylation of a subset of consensus sites.…”

“…5 B, lane a). The predominant glycoform of invertase secreted by wild-type cells contains, on average, 9-10 N-linked oligosaccharides (Reddy et al, 1988). Mature invertase migrates as a 100-150-kD smear due to extensive elongation of core oligosaccharide chains in the Golgi apparatus (Esmon et al, 1981).…”

Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins

“…5 C ). Extensive mannosylation of the N-linked oligosaccharides of invertase in the Golgi ( 45 ) yields a diffuse high–molecular weight smear (Suc2 Golgi). Endo H digestion confirmed that the immunoprecipitated products identified as Suc2 Golgi and Suc2 ER contain N-linked glycans.…”

“…5C). On average, ten of the thirteen glycosylation acceptor sites in invertase are modified upon J o u r n a l P r e -p r o o f translocation of Suc2p into the ER lumen (45) giving rise to the heterogeneous cluster of bands labeled as the ER form of Suc2 (Fig. 5C).…”

Paleoproteomics explained to youngsters: how did the wedding of two-dimensional electrophoresis and protein sequencing spark proteomics on: Let there be light

“…This was however the first method able to analyze large and polar molecules such as peptides [72], but half a decade was needed for more practical methods to be applied for peptide ionization [73][74][75]. So at the end of the 80's both mass spectrometry and Edman sequencing had around the same performances in sensitivity and in throughput, with the incredible advantage for mass spectrometry of being able to analyze modified amino acids [76,77] even with non classical modifications [78]. However, reading a sequence by mass spectrometry required interpreting manually the MS/MS fragmentation spectra [79,80] a process that required as much expertise as running an Edman sequencer.…”