Phosphorylation Analysis Of Num1 And Identification Of Its Interacting Proteins

During the mitosis in eukaryotes,the spindle must be properly oriented to ensure that the fate determinants can be accurately divided into every daughter cell.The cytoplasmic dynein plays a key role during spindle orientation in various types of cells.Dynein needs to be anchored at the cell membrane in order to produce pulling forces to move the spindle while dynein itself moves along the microtubules.In budding yeast,dynein is associated to the cell membrane through the membrane protein Num1.Num1 is also the only factor involved in spindle orientation that has been found thus far to localize to the cell membrane.It is composed of multiple domains.The PH domain proximal to its C-terminus mediates its binding to the cell membrane,while the PA domain at its N-terminus mediates the formation of multiple molecule complexes of Num1 through the interaction with itself.The PA domain also mediates the interaction of Num1 with dynein.Besides its role in spindle orientation,Num1 also mediates the attachment of mitochondria with the cell membrane.However,it is not clear how the activity of Num1 is regulated during these processes.We set out to study Num1 in this study from two aspects: 1)to analyze the phosphorylation of Num1;2)to identify regulators of Num1 through isolating proteins that interact with the PA domain.Our sequence analysis showed that there are a series of potential phosphorylation sites in Num1,in particular,at its C-terminal sequence.We analyzed the phosphorylation of MMNum1 composed of the C-terminal sequence and the PA and PH domains using phos-tag SDS-PAGE technique.MMNum1 tagged with Myc or GFP migrated as multiple bands in phos-tag SDS-PAGE,indicating a modification of phosphorylation.In addition,treatment with a protein phosphatase removed the slowly migrating band,further suggesting the occurrence of phosphorylation in MMNum1.What is more,removing the C-terminal sequence of MMNum1 decreased the accumulation of phosphorylated bands.It is therefore speculated that amino acids residing at its C-terminus are involved in the phosphorylation of MMNum1.In our experiments to identify proteins interacting with Num1,we expressed the PA domain in E.Coli cells,purified recombinant proteins of the PA domain,and used them for pull-down experiments in combination with spectral analyses.We successfully identified a number of important new proteins that interact with Num1.Based on their functions,they can be classified as the following groups: 1)ribosomal proteins;2)subunits of the anaphase promoting complex(APC)that regulates protein degradation during the cell cycle;3)members of the eisosome complex that associates with the cell membrane;4)ER and Golgi proteins involved in protein folding,sorting and transportation;5)nuclease and protein enzymes functioning in gene transcription and translation;and 6)other proteins with various functions.Confirmation of the phosphorylation and identification of interacting proteins of Num1 laid a good foundation for us to unravel the mechanism of Num1 function during spindle orientation and in mitochondria.