The Study Of Localization And Functions Of Transmembrane Protein MS9 On Exosomes

As a classical type of integral membrane proteins,transmembrane protein spans the entirety of the cell membrane to which it is permanently attached and also plays crucial roles in the physiological functions of cells.Transmembrane proteins may go through a significant conformational change to transport specific molecules and ions;or promote signal exchange between cells through the interaction with downstream messengers.All cells can secrete various types of extracellular vesicles,and this process is conserved throughout evolution from bacteria to plants and humans.Based on their biogenesis,extracellular vesicles are classified into three groups:exosomes,microvesicles and apoptotic bodies.Exosomes and microvesicles are secreted by living cells,which were previously considered as a mechanism to dispose of cellular components but have gradually drew more attention in recent years.At present,many studies have shown that exosomes contain nucleic acids and proteins so that they have capability to exchange components between cells.Therefore,they may act as signaling vehicles in normal cell homeostatic processes or as a consequence of pathological developments.Clinical application indicates that exosomes are abundant and stable sources of circulating biomarkers.The previous study in our laboratory have shown that a new transmembrane protein MS9 can interact with Smad ubiquitin regulatory factors 1(Smurf 1).Currently,there are few studies related to MS9.The protein structure,cell localization and biological functions of MS9 remain to be explored.In this study,we found that transmemb rane protein MS9 may localize on plasma membrane and some vesicles.We fiurther confirmed that these vesicles are early endosomes by examine the colocalization and interaction of MS9 and EEA1,a marker protein of early endosome.In addition,we also purified exosomes by ultracentrifugation and revealed that exosomes also contain MS9 protein.Using florescent protein tag labeling and NanoFCM technique,we identified that MS9 cross the vesicle membrane with its C-terminus left outside.Moreover,we treated MDA-MB231 breast cancer cells with exosomes containing MS9 and found that MS9 could be transferred into the cells and inhibit the phosphorylation of EGFR.