Preliminary Study On The Functions Of Num1 Interacting Proteins Pil1 And Msp1 In Saccharomyces Cerevisiae

In multicellular organisms,communication and signal transmission between cells are very important for cell growth,division,death and differentiation.The direct contact between cells is a way for cells to transmit signals.There is a similar way of communication within the cell,namely the inter-organelle contact.In budding yeast,the membrane protein Num1 is the key tether of MECA(mitochondria-endoplasmic reticulum-cortex anchor),which bridges the contact between mitochondria and other organelles,and also mediates the division and localization of mitochondria.Meanwhile,Num1 mediates the correct positioning of the spindle in mitosis to ensure that the nuclei are accurately distributed to each daughter cell.Therefore,it is necessary to study the organelle contact involving Num1 to explore how the organelle contact coordinates the function of each organelle.A series of proteins that may interact with Num1 were identified through pull-down experiments and LC-MS(Liquid chromatography-mass spectrometry).In this study,Msp1,which is also a mitochondrial outer membrane protein like Num1,and Pil1,which shows similar localization to Num1 to the cell membrane,were chosen to study,focusing on confirming their interaction with Num1,and whether they are functionally relevant to Num1 in mitochondria and during spindle positioning.The major work and results are as follows:1.To confirm the interaction between Pil1 and Num1 proteins,yeast strains for co-immunoprecipitation experiment were constructed,including YT47(GAL1-3HA-PIL1),YT52(GAL1-NUM1-YFP)and YT84(GAL1-3HA-PIL1 GAL1-NUM1-YFP),and co-immunoprecip experiment was performed.The GAL1-Num1-YFP protein was precipitated with a GFP antibody,and the co-immunoprecipitated GAL1-3HA-Pil1 was detected.However,the protein sample that did not express GAL1-Num1-YFP was found to bind the GFP antibody along with the magnetic beads unspecifically.Therefore,the experiment conditions or reagents need to be changed for further experiments.2.To examine the effect of deleting PIL1 on spindle positioning and in turn on nuclear migration,the pil1? strain was constructed,and its frequency of producing binucleated cells was counted.The frequency of binucleated cells was observed to be 1.72% for the pil1? strain,much lower than that of the num1? strain,which is 22%,suggesting that Pil1 might not be a key factor regulating spindle positioning.3.To confirm the interaction between Msp1 and Num1 proteins,yeast strains for co-immunoprecipitation experiment were constructed,including YT106(GAL1-MSP1-3HA),YT112(GAL1-MSP1-3HA GAL1-NUM1-YFP),and YWL490(GAL1-NUM1-YFP).But problems in co-immunoprecipitation experiments similar to those of the co-immunoprecipitation experiments of Pil1 and Num1 were encountered.4.To examine the role of MSP1 in nuclear migration,the msp1? strain was constructed,and its frequency of producing binucleated cells was counted.The frequency of binucleated cells was observed to be 0.9% for the msp1? strain.Combined with the localization of Msp1 to mitochondria,it is speculated that Msp1 may not be involved in spindle positioning.5.To determine the functional relevance of Msp1 with Num1,the msp1? NUM1-GFP strain was constructed.The fluorescent microscopic observation indicated that Num1-GFP still localized to the cell membrane in the absence of MSP1.But whether the fluorescent intensity of Num1-GFP clusters is affected remains to be determined.In addition,the MSP1-3mCherry NUM1-yEGFP strain was constructed in order to observe the co-localization of Msp1 and Num1.6.To determine the functional relevance of Msp1 with Num1 in mitochondria,single or double mutants of msp1?,get1? and get2? expressing COX4-mCherry were constructed for observing the morphology of mitochondria and comparing with the num1? strain.It was observed that the morphology of mitochondria in msp1? and get1? single mutants was similar to that of wild type cells.However,the mitochondria in the msp1? get1? double mutant showed fragmentation,consistent with reported results.Detailed comparison of mitochondrial morphology between msp1? get1? and num1? cells can be conducted in the future.These results lay the foundation for uncovering the effect of organelle contact on cell growth and function and for studying the mechanism of inter-organelle contact.