Abstract
Insulin stimulates glucose transport by promoting translocation of intracellular glucose transporter-4 (GLUT4)-containing vesicles to the cell surface of adipose cells. Although numerous studies have examined the insulin signaling mechanisms leading to translocation of GLUT4 vesicles to the plasma membrane, this process is still unclear. In this study, we used mass spectrometry in combination with subcellular fractionation to identify protein components of THE plasma membrane in 3T3-L1 adipocytes. The cells were homogenized and subjected to a series of centrifugation procedure to prepare plasma membrane and low and high density microsome fractions. Proteins were identified by SDS-PAGE and in-gel digestion, followed by tandem mass spectrometry. All MS/MS spectra were searched using MASCOT program and the results were processed and verified by our originally developed software. Using this approach, we have identified about 500 proteins, 55% of which had transmembrane domains, and which contained high molecular weight proteins over 100kDa. In addition, we investigated plasma membrane proteins after insulin stimulation and compared with those from the basal condition. Insulin-responsive amino peptidase (IRAP), which is known as one of the resident proteins in GLUT4 vesicle, was used to evaluate mass spectrometric comparison. The peak intensity of tryptic fragments of IRAP increased after insulin stimulation in the plasma membrane, which was also confirmed by western blotting. We have found a novel protein, which increased in the plasma membrane fraction after insulin stimulation.
