Dom34 Links Translation to Protein O-mannosylation.

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5'-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3'-UTR of transcripts.

Damage to the glycoshield activates PMT-directed O-mannosylation via the Msb2-Cek1 pathway in Candida albicans.

Protein-O-mannosyltransferases (Pmt) transfer mannosyl residues to secretory proteins. Five isoforms of Pmt proteins in the human fungal pathogen Candida albicans have distinct functions in growth, morphogenesis and antifungal resistance. We found that PMT genes encoding the major isoforms Pmt1, Pmt2, Pmt4 are regulated differently in response to impaired glycostructures. While the PMT1 transcript level increased in cell wall mutants and under inhibition of N-glycosylation by tunicamycin, PMT2 and PMT4 transcripts were upregulated only by inhibition of Pmt1 activity. Reporter fusions revealed specific promoter sequences to be required for PMT1 repression in undamaged cells, which was de-repressed by tunicamycin. Constitutive PMT1 de-repression was observed in mutants lacking the Cek1 MAP kinase and its upstream sensor Msb2. In contrast, in msb2 and cek1 mutants, upregulation of PMT2/PMT4 by Pmt1 inhibition did not occur and basal expression of both transcripts were decreased. We identified Ace2 as a novel transcription factor, which upregulates PMT basal expression and induction in response to glycostructure damage. Mutants lacking Msb2, Cek1 and Ace2 were supersensitive to glycosylation and cell wall inhibitors. We propose that a Msb2, Cek1 and Ace2 signalling pathway addresses PMT genes as downstream targets and that different modes of regulation have evolved for PMT1 and PMT2/PMT4 genes.

Transcriptional and physiological adaptation to defective protein-O-mannosylation in Candida albicans.

Five Pmt isoforms O-mannosylate secretory proteins in Candida albicans. Comparisons of genome-wide transcript patterns of each pmt mutant revealed commonly downregulated genes involved in glycolysis and glycerol production. Increased phosphorylation of the Cek1p- but not the Mkc1p-MAP kinase, as well as increased transcript levels for some stress-related genes were detected in the pmt1 strain but not in the other pmt mutants. The transcriptomal pattern after short-term inhibition of Pmt1p activity confirmed stress responses, but did not indicate an alteration of glycolytic flow. Short- but not long-term adaptation to Pmt1p inhibition required signalling components Cek1p, Mkc1p, Efg1p and Tpk1p. Cna1p (calcineurin) but not its downstream effectors Crz1p and Crz2p was generally essential to allow growth during Pmt1p inhibition; accordingly, cyclosporin A strongly inhibited growth of the pmt1 mutant. The lack of Pmt isoforms influenced transcript levels for the remaining isoforms both positively and negatively, suggesting complex cross-regulation among PMT genes. These results confirm individual functions of Pmt isoforms but suggest a common biphasic adaptation response to Pmt deficiency. While known signalling pathways modulate adaptation for a short-term, long-term adaptation requires calcineurin, adjustments of remaining Pmt activities and of glycolytic flow.