Truncations and functional carboxylic acid residues of yeast processing alpha-glucosidase I.
Yeast alpha-glucosidase I (Cwh41p) encoded by CWH41 is an endoplasmic reticulum (ER) membrane-bound glycoprotein (833 residues), which plays an important role in the early steps of the N-glycosylation pathway. In this study functional expression of three truncated fragments of Cwh41p, all containing the catalytic region, was investigated. Cwht1p (E35-F833), with deletion of the N-terminus and transmembrane domain, was expressed as a catalytically active fragment while R320-F833(Cwht2p) and M526-F833 (Cwht3p) were not detected. Significantly higher glucosidase I activity was found in a soluble extract from yeast overexpressing CWHT1 (1,400 U/g biomass) than yeast overexpressing CWH41 (300 U/g biomass). Cwht1p was purified as a soluble 94 kDa non-glycosylated protein with a specific activity (3,600 U/mg protein) comparable to that of the soluble alpha-glucosidase I (3000 U/mg protein). These findings indicate that the active conformation of the enzyme is not dependent on protein glycosylation and suggest that the M1-I28 region of Cwh41p carries an ER-targeting signal sequence. In addition, two highly conserved carboxylic acid residues, E580 and D584 of Cwht1p (corresponding to E613 and D617 of Cwh41p), located within the catalytic domain of yeast enzyme were subjected to mutation. Substitution of each residue with Ala resulted in low expression and undetectable glucosidase I activity. These findings indicate that E613 and D617 play a crucial role in maintaining alpha-glucosidase I activity.
Faridmoayer
Amirreza
A
Food, Nutrition, and Health Program, University of British Columbia, Vancouver, V6T 1Z4, Canada.
Scaman
Christine H
CH
D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D021382:Protein Sorting Signals; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000520:alpha-Glucosidases
D002264:Carboxylic Acids; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D010455:Peptides; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; C098060:CWH41 protein, S cerevisiae; D000520:alpha-Glucosidases