Mechanism Of PMT1 Deficiency Extending The Replicative Lifespan In Saccharomyces Cerevisiae

Protein-O-mannosyltransferase (PMT) family has seven members, which are evolutionarily conserved from yeast to mammals. They locates in endoplasmic reticulum (ER) of yeast, and involve in ER homeostasis by regulation of ER unfolded protein response (UPR). The ER stress sensor Irelp acts through the ER-stress responsive transcription factor Haclp to regulate transcription of UPR target genes, and these upregulated genes enhance the protein folding capacity and alleviate ER stress. During the process, HAC1 mRNA splicing by Irelp is a key step to the activation of UPR pathway, and is commonly used to evaluate UPR activity. The UPR and ER stress have been implicated in aging and age-related disease in several organisms.Methods:1. PMT family gene deletion (pmtl△～pmt6△) strains, PMT I and IRE1 or HAC1 double-gene deletion (pmtl △hacl△ and pmtl△irel△) strains were successfully constructed through homologous recombination. The physical separation of buds from yeast mother cells was achieved using a manual micromanipulator equipped with a fibre-optic needle under optical microscope (Axio Scope. Al, Carl Zeiss). The replicative lifespan (RLS) datasets from all buds were analyzed using a Wilcoxon Rank-Sum test. We explored the role of PMT family members in lifespan modulation. Moreover, HAC1 mRNA splicing in pmtl△ strain was analyzed by RT-PCR and quantified by densitometry, and transcription levels of canonical UPR target genes were detected by quantitative RT-PCR and expression level of ER chaperone protein Kar2 was detected by Western Blot for exploring the relationship between PMT1 regulating RLS and UPR pathway.2. TED1 deficiency (tedl△) strain, TED1 and PMT1 double-gene deletion (pmtl△tedl△) strain (Pmtlp forms complex with Tedlp), and three-gene deletion (pmtl△tedl△hacl△) strain were successfully constructed through homologous recombination. We analyzed these mutant strains’lifespan and UPR activity by detecting the RLS and quantitative RT-PCR, and investigated the relationship for PMT1 and TED I modulating the lifespan. We also explored the mechanism of UPR pathway modulating the lifespan of yeast.3. Tunicamycin and Congo red/Calcofluor White Stain are ER stress inducer and cell wall stress inducers, respectively. The sensitivity of PMT1 and TED1 deficiency strains to ER stress and cell wall stress were analyzed by colony-fonning ability. We investigated the relationship for the cell stress reponses and UPR activity or cell wall integrity (CWI) pathway, and further explored the relationship between the RLS and cell stress responses.Results and conclusions:1. Among all the strains, only pmtl△ strain had an increased lifespan (about 20%, P<0.0001), and deficiency of IRE1 or HAC shortened longevity of pmtl△ strain. Deficiency of PMT1 induced the higher expression levels of spliced HACl mRNA (approximately 29.5%) and the canonical UPR downstream target genes, including genes involved in oxidative folding (EUG1 and ERO1), protein trafficking (FKB2), and chaperone function (KAR2 and LHS1). Similar to the quantitative PCR data, the protein levels of Kar2p were obviously increased in the pmtl△ strain relative to wild type (WT). The results indicated that PMT1 deficiency extended the RLS of yeast, and the UPR activity was involved in the longevity of the pmtl△ strain.2. The mean lifespan of the tedl△ strain decreased (about 17%, P<0.001) as compared with WT. Deficiency of PMT1 significantly extended the RLS of tedl△ strain, which was depended on HAC1. Deficiency of PMT1 induced the higher expression levels of spliced HAC1 mRNA and the UPR target genes. The results indicated that PMT1 deficiency rescued the lifespan of ted1△ strain through the UPR signaling pathway.3. The pmt1△ strain was sensitive to ER stress. Deficiency of PMT1 enhanced the hacl△ strain’resistance ability to ER stress. Deficiency of PMT1 enhanced the tedl△ strain’s sensitivity to ER stress and cell wall stress. The results indicated that the longevity yeast strains might not have the enhanced resistance ability to stress.In conclusion, deficiency of PMT1 extended lifespan of wild type yeast cell and tedl△ strain, which was related with the UPR activity. Although the deficiency of PMT1 can induce the increased expression of the genes involved in UPR and CWI signaling pathway, the yeast strains didn’t displayed the enhanced resistance ability to stress. The results indicated that further studies are required for investigation the relationship between the RLS and stress response.