Glycerol Transporter 1 (GT1) And Zinc-regulated Transporter 1(ZRT1) Synergize For Zinc Homeostasis In Pichia Pastoris

Pichia pastoris is a type of methanol-nutrient yeast that can use methanol as the sole carbon source and energy source.It contains two alcohol oxidase genes,aox1 and aox2,which are strongly methanol-inducible.Because P.pastoris has a high growth rate,it can grow in a simple,inexpensive medium,suitable for small-scale and large-scale production.Having the post-translational modification mechanism of eukaryotic expression system,it is widely used in recombinant DNA technology to produce proteins.The gt1?GenBank:CP014715.1?gene encodes a membrane transporter responsible for glycerol uptake in P.pastoris.The zrt1?GenBank:CP014717.1?mRNA level was highly increased in the P.pastoris GS115?gt1 mutant according to previous studies,which suggested that GT1may be similar to ZRT1 in function.First,according to NCBI analysis,the P.pastoris zrt1 gene was predicted to be similar to the high-affinity zinc transporter gene?GenBank:CP029160.1?from Saccharomyces cerevisiae.HMMTOP transmembrane segment analysis showed that GT1 had twelve hydrophobic domains and ZRT1 has seven hydrophobic domains.TMPres simulated the topology structures of the two proteins and found that they all had an intracellular loop located between the middle transmembrane segments.There were typical metal binding motifs in the loop,such as histidine motif,cysteine motif.These sites can help protein bind to substrate Zn²⁺and execute transport function.Based on the protein properties of the two,overexpression strains,delletion strains,and RFP-fused expression strains of GT1 and ZRT1 were constructed,respectively,and their Zn²⁺equilibrium dissociation constants,Zn²⁺consumption levels,mRNA levels,translation levels were detected.The subcellular fluorescence localizations of RFP-fused proteins in vivo were observed.Consistent with their predicted function,both proteins could bind zinc in vitro.The binding affinity of ZRT1 to zinc?6.63?M?was higher than GT1?16.4?M?.Further determined by the subcellular fluorescence localizations of the RFP-fused proteins,both were located to the P.pastoris cell membrane.The zinc consumption levels of the delletion strains?gt1,?zrt1,?gt1?zrt1 were detected by Zn²⁺fluorescence quantitative method to verify the Zn²⁺uptake capacity of GT1 and ZRT1.The zinc concentrations in the extracellular medium during the growth of the deletion strains were detected,and it was found that the deficiency of these two proteins impaired zinc uptake activity of cells in zinc-limited media.Furthermore,high zinc induced down regulation in transcription level and protein level of zrt1,and resulted in ZRT1 internalizing into the vacuole;Gt1 only responded to zinc regulation in glycerol.GT1 was subjected to glucose-induced inactivation.Thus,GT1 and ZRT1 could synergistically maintain zinc homeostasis in glycerol,while the block of GT1 function might lead to the upregulation of zrt1 in glucose.These results indicate that GT1 and ZRT1 are regulated by different regulatory mechanisms,and they transport zinc or Zn-ligand under specific conditions to maintain zinc homeostasis.This study provided new ideas for exploring the function of GT1,and successfully identified a new function of GT1,and conducted a preliminary exploration for the metal cofactor effect in glycerol suppression.At the same time,it provided a reference for the functional study of eukaryotic membrane proteins in vitro and lay a foundation for the structure research of membrane protein GT1.