Metal Sensitivity Caused By Deletion Of High-affinity Ferroportin In Schizosaccharomyces Pombe And Its Effect On Mitochondrial Function

There are iron transport systems with high affinity and low affinity in Schizosaccharomyces pombe.The high affinity iron transport system mainly includes iron reductase Frp1,polycopper oxidase Fio1 and percolase Fip1.Frp1 protein can reduce Fe³⁺to Fe²⁺,or Cu²⁺to Cu⁺.These reduced metal ions are then oxidized to oxidized metal ions by Fio1,and finally these metal ions are infiltrated into the cell by peroxidase Fip1.Low-affinity iron transporters include Fet4 and Pdt1,which can transport divalent iron ions or other transition metal ions into cells.In the case of sufficient iron,the low affinity iron transport system plays a role,while in the case of iron deficiency,the high affinity iron transport system plays a role.In Schizosaccharomyces pombe,the sensitivity of transition metal ions caused by high affinity iron transporter deletion and its effect on mitochondrial function have not been reported.This paper is mainly studied from these two aspects.Firstly,the protein domains of iron transport system with high affinity and low affinity were analyzed by bioinformatics.It is known that Fio1 protein has a conservative polycopper oxidase domain and Frp1 protein has a conservative iron reductase domain.Through sequence alignment of homologous protein domains,it is found that the histidine of these two domains is conservative,which is involved in the binding of metal ions.Fet4 protein has a conserved low affinity iron permease domain and Pdt1 has a conserved Nramp domain.Through homologous protein domain alignment,it is found that their aspartic acid and glutamic acid are highly conserved.Studies have shown that negatively charged amino acids play an important role in binding to metal ions,so Fet4 and Pdt1 play a role in transition metal transport.Phenotypic experiments showed that the high affinity iron transport gene fio1deletion strain showed growth defects on the iron deficient plate,while the low affinity iron transport gene deletion strain grew normally.After the loss of high affinity iron transporter,the sensitivity to transition metal ions increases,which is speculated to be caused by the accumulation of transition metal ions caused by the increase of the activity of low affinity iron transporters.Then we constructed double knockout strains and triple knockout strains with high affinity and low affinity,and found that some double knockout strain could save the sensitivity of transition metal ions caused by high affinity loss.At the same time,we detected the RNA levels of fet4and pdt1 in the medium of transition metal ions at the later stage of cell growth,and found that the transcription levels of fet4 and pdt1 of wild-type y HL6381,fio1knockout strain were significantly lower than those of wild-type.We infer that cells may gradually resist the accumulation of transition metal ions through some mechanism.Secondly,we found that fio1 and frp1 deletion strain showed growth defects on the non-fermented medium with glycerol as the only carbon source.By observing their mitochondrial morphology by fluorescence microscope,we found that the mitochondrial morphology of frp1 deletion strain showed fragmentation.It is speculated that the deletion of fio1 and frp1 genes may lead to the impairment of mitochondrial respiratory chain function.Next,we explored the changes of RNA and protein levels encoded by the mitochondrial genome to explain the impairment of mitochondrial function,but only found that in frp1 deletion strain,the expression of Cox1 and Cox3 proteins decreased partially,Cob1,Atp6 and Cox2 did not change,and the RNA levels of these genes did not change.The reasons for the impairment of mitochondrial function need to be further explored.This paper mainly consists of two aspects:one is that the loss of high affinity iron transporter in Schizosaccharomyces pombe will cause the sensitivity of transition metal ions,and some double knocking strain can save the sensitivity of transition metal ions caused by the loss of high affinity.Second,the deletion of high affinity iron transporter can affect the function of mitochondria.The studies on these two aspects provide a theoretical basis for the future exploration of high affinity iron transport system in S.pombe.