Identification of the archaeal alg7 gene homolog (encoding N-acetylglucosamine-1-phosphate transferase) of the N-linked glycosylation system by cross-domain complementation in Saccharomyces cerevisiae

Abstract

The Mv1751 gene product is thought to catalyze the first step in the N-glycosylation pathway in Methanococcus voltae. Here, we show that a conditional lethal mutation in the alg7 gene (N-acetylglucosamine-1-phosphate transferase) in Saccharomyces cerevisiae was successfully complemented with Mv1751, highlighting a rare case of cross-domain complementation.

FIG. 1.

Multiple sequence alignment with hierarchical clustering (7) of S. cerevisiae and M. voltae GPTs. Black boxes indicate identical amino acid residues. The conserved Asp-Asp in the putative DDXXD motif (marked with asterisks) is predicted to be coordinated to the Mg²⁺ cofactor. The putative active site nucleophilic Asp VFPGDT motif for Mv1751 is indicated with rectangles. •, conserved lysine located downstream of the DDXXD motif.

FIG. 2.

Rescue of YPH499-HIS-GAL-ALG7 by complementation with either the human ALG7 or Mv1751 gene. The conditional lethal mutant YPH499-HIS-GAL-ALG7 was transformed with the pRS426Met plasmids carrying either the human ALG7 (HsALG7) or the archaeal Mv1751 (MvALG7). The transformed cells were then streaked onto plates containing minimal medium lacking histidine and containing either galactose (II) or glucose (III) and incubated at 30°C. Panel III clearly shows that both the human and archaeal alg7 genes rescue the conditional lethal phenotype of YPH499-HIS-GAL and complement the yeast alg7.

FIG. 3.

Indirect immunofluorescence microscopy comparing the distributions of the ER marker BiP and the expressed Mv1751 in S. cerevisiae. FLAG-tagged Mv1751 is clearly visible inside cells containing the MvALG7 plasmid, while cells containing the empty pRS426Met plasmid showed no fluorescence. Costaining with BiP showed nearly complete overlapping with Mv1751. Cells were stained with DAPI (4′,6′-diamidino-2-phenylindole) to detect chromosomal DNA. PC, phase contrast.