A hypomorphic allele of the ALG7 gene causes mtDNA instability and respiratory deficiency in Saccharomyces cerevisiae

In eukaryotes, modification of proteins at asparagine residues with oligosaccharides (N-glycans) is required for a plethora of developmental and physiological processes. All N-glycans originate from a common lipid-linked oligosaccharide (LLO) precursor, whose synthesis is initiated by the dolichol-phosphate-dependent N-acetylglucosamine-1-phosphate transferase (GPT) encoded by the essential ALG7 (Asparagine Linked Glycosylation gene 7) gene. Inappropriate reduction of ALG7 expression in yeast cells, generated through sporulation of a heterozygous ALG7/alg7 diploid, gives rise to altered phenotypes including cell cycle arrest and aberrant morphology. To further explore the effects of ALG7 dosage on cell function, mutants with a genomic hypomorphic allele of ALG7 were constructed by directly integrating a plasmid, containing a partial deletion in the 3′ UTR (untranslated region) of the ALG7 gene, into the ALG7 locus in two genetically distinct mitotic yeast cells. We show that both alg7 mutants exhibited decreased ALG7 mRNA and GPT activity levels, lower abundance of LLO pools and under-glycosylation of carboxypeptidase Y (CPY), albeit to different extents. Unlike the alg7 sporulation mutants, however, neither of the alg7 vegetative mutants was able to grow on glycerol, and this defect could not be rescued by the wild type ALG7 gene. DNA array analyses showed that alg7 vegetative mutants lacked any measurable expression of mitochondrial (mt) genes. Accordingly, the alg7 mutants were negative for mtDNA staining with DAPI (a DNA binding UV fluorescent dye), although they contained mitochondria as documented by DiOC₆ (fluorescent dye that illuminates active mitochondria) fluorescence. Notably, neither mutant exhibited respiratory activity. Our studies show that ALG7 knockdown in vegetative yeast cells induces mtDNA instability and respiratory deficiency. This study describes the first N-glycosylation mutants classified as rhoo petites. To determine whether the effect of ALG7 on respiration reflects the role of N-glycosylation in cellular ATP metabolism an alg6 mutant, with a less severe LLO defect, was studied and it too exhibited stress-induced respiratory deficiency. These studies shed light on the interactions between N-glycosylation and cellular metabolic circuitry and will further our insights into the pathobiology of congenital disorders of glycosylation (CDGs) in humans.