Abstract
It is accepted that glycosyltransferase-generated nucleoside diphosphates are converted to monophosphates in the secretory pathway by nucleoside diphosphatases (NDPases) to provide substrates for antiport transport systems by which entrance of nucleotide sugars from the cytosol into the lumen is coupled to exit of nucleoside monophosphates. Working with Saccharomyces cerevisiae mutants affected in anterograde and/or retrograde endoplasmic reticulum (ER)-Golgi vesicular traffic and/or defective in one or both secretory pathway (Golgi) NDPases, we show that UDP-Glc: glycoprotein glucosyltransferase-mediated glucosylation is not dependent on the presence of NDPases or on ER-Golgi vesicular traffic and that GDP-Man-dependent N- and O-mannosylations are reduced but not abolished in the absence of NDPases in the secretory pathway. Further, the absence of the main Man-1-P transferase (a Golgi GMP-generating enzyme) does not modify the limited mannosylation observed in the absence of NDPases. Based on these results and on available additional information, we suggest that in the absence of NDPases, the already characterized nucleotide sugar transporters allow entrance of nucleotide sugars into the luminal compartments and that resulting nucleoside diphosphates exit to the cytosol by a still unknown mechanism. Further, an unexpected side result suggests that formation of Ser/Thr-Man(2) may occur in the ER and not exclusively in the Golgi.
Highlights
Introduction of Unnatural Amino Acids viaRibosomal Protein Synthesis—Liu and Schultz [25] have designed methods to overcome the limitations set by the genetic code and added ϳ70 novel amino acids with diverse side chains to enrich protein functions
Cloning a Variant of hGST M2-2 Optimized for Chemical Modification—The three Cys residues in wild-type hGST M2-2 were replaced with Ala, and the enzyme variant C87A/C115A/ C174A was further modified by a M212C mutation to give mutant C87A/C115A/C174A/M212C
A close-up of the hGST M2-2 active site displays Met-212 located near the C terminus and situated between the catalytically significant Tyr-116 and Ser-210 (Fig. 1a)
Summary
Introduction of Unnatural Amino Acids viaRibosomal Protein Synthesis—Liu and Schultz [25] have designed methods to overcome the limitations set by the genetic code and added ϳ70 novel amino acids with diverse side chains to enrich protein functions. Post-translational Modifications of Proteins—In part, the need for other functions of the amino acids has been solved by nature via post-translational modifications [1]. This process is restricted to certain amino acids, and the chemistries that are introduced are functionally limited. A different approach to the generation of unnatural amino acids involves the chemical modification of Cys ex vivo, as developed in this work. The chemical modification is usually expeditious and can be performed with native protein or under denaturing conditions, depending on the accessibility of the Cys residue. The method applies without difficulty only to Cys residues, this intrinsic limitation is offset by the rich chemistry of thiol modifications
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