Functional Characterization Of The Yeast Vac14 Homologous Protein VacA In Aspergillus Nidulans

Phosphatidylinositol 3-Phosphate and phosphatidylinositol 3,5-bisphosphate are important lipid small molecular substances in eukaryotic cells.They regulate the circulation,fusion,cytosis,autophagy and ion transport of the membrane in the eukaryotic cells.In yeast cells,Vac14 forms a complex with kinase Fab1 and phosphatase Fig4,which is responsible for the synthesis of phosphatidylinositol-3,5-bisphosphate(Ptd Ins(3,5)P₂)and the transformation of phosphatidylinositol-3-phosphate(Ptd Ins3P).In this process,Fab1 phosphorylates Ptd Ins3p to produce Ptd Ins(3,5)P₂,which phosphatase Fig4 dephosphorylates Ptd Ins(3,5)P₂and reduces it to Ptd Ins3p,while Vac14 regulates this dynamic process as a scaffold protein to provide a site for this reaction,and fix this reaction in a specific space to make the reaction more sufficient.Previous studies have found that in Saccharomyces cerevisiae,knockout of Vac14 leads to a significant decrease in the level of Ptd Ins(3,5)P₂,which makes the cells unable to withstand osmotic stress,and then makes the vacuoles significantly larger.In the human body,Vac14 mutation will cause lysosomal function damage,as well as abnormal endosome transport,and then lead to a series of diseases,resulting in patients with mental degradation,impaired behavior and other symptoms.In filamentous fungi Aspergillus nidluans,we found that Vac14 also had homologous protein with the gene number AN10682 and protein similarity is 37.65%,and the predicted protein domain was very similar,but the relevant cytological function has not been reported.According to the nomenclature rule of Aspergillus nidulans,it was named Vac A.In this paper,we comprehensively studied the biological function of Vac A by means of genetics,biochemistry and cytological morphology observation,and studied the potential interaction protein network of Vac A by labeling Vac A protein and protein binding experiment in vitro.The results are as follows:the whole gene of vac A was knocked out by homologous recombination gene replacement,and the culture phenotype analysis showed that the deletion of Vac A led to the decrease of sporulation,and high temperature and high concentration of spore inoculation density would aggravate the decrease of sporulation phenotype.In addition,knockout of Vac A made the strain more sensitive to the cell wall destruction reagent CR(Congo Red),and the knockout strain showed serious growth defects in the medium added CR.This phenotype is consistent with the phenotype of mid A and cch A mutations which are belong to high-affinity calcium influx system,suggesting that vac Ais involved in the regulation of intracellular calcium balance.The results showed that the sporulation ability of the knockout strain could be greatly restored by adding calcium into the medium.The intracellular matrix and vacuoles of important calcium storage organelles were measured by using aequorin(AEQ).The results showed that compared with the wild parent strain,the cytosolic calcium concentration of the knockout strain was decreased,while the vacuolar calcium concentration was increased,but there was no significant difference in the transcription level of calcium pump related genes.Through GFP labeling and RFP labeling of related proteins,it is found that Vac A is located on the cell matrix and vacuolar membrane respectively,which is similar to the location of Vac14 in yeast and humans.Further in vitro protein binding experiments(Pull-down)conducted a comprehensive mass spectrometry detection of the interaction protein complex of Vac A,and found some proteins related to phospholipid synthesis and proteins related to calcium ion transport.It is suggested that Vac A can regulate the intracellular calcium ion balance by interacting with these proteins.The molecular mechanism of specific regulation needs to be further developed in the future.This study analyzed the biological function of Vac A in Aspergillus nidulans.The important function of Vac A in the calcium ion balance and the interaction protein network found in the study also have certain reference value for clarifying the functions of Vac A homologous proteins of other species.