Insulin homeostasis in pancreatic -cell is now recognized as a critical element in the progression of obesity and type II diabetes (T2D). diabetes (T2D) (Ashcroft and Rorsman, 2012), it is crucial CX-4945 to understand the protein interactions that govern the biogenesis and release of insulin into plasma. Orci and colleagues initially described the trafficking itinerary of insulin through the secretory pathway of the pancreatic -cell using electron microscopy (Orci et al., 1986; Orci et al., 1985). Mature insulin is synthesized in the ER, transported through the secretory pathway and then stored in two functionally distinct pools of granules: the reserve pool (RP) and the readily releasable pool (RRP) from which insulin is released in a biphasic fashion upon stimulation with glucose (Seino et al., 2011). We now know that folding in the ER, ER-to-Golgi transport and secretion of proteins to the extracellular milieu in response to stimulus is aided and monitored by the proteostasis network that make continual adjustments to the protein fold (Balch et al., 2008; Hutt and Balch, 2013; Powers and Balch, 2013). In human disease, early components of the folding machinery play a critical role in -cell biology (Back and Kaufman, 2012). Surprisingly, little is known about the proteins that interact with insulin directly or indirectly to promote conformational maturation required to maintain the biphasic insulin release that is defective in T2D. To explore the proteins promoting insulin synthesis and folding, granule maturation and secretion, previous proteomics studies focused on whole CX-4945 human pancreatic islets under basal (Waanders et al., 2009) and elevated CX-4945 glucose levels (Schrimpe-Rutledge et al., 2012). Using mass spectrometry of purified intact islets (which contain multiple cell types), these studies identified a large number of metabolic and signaling pathway components. Studies using insulin granules purified from a rat insulinoma cell line, INS1-E, by subcellular fractionation with or without immuno-affinity purification of the granule surface-associated protein VAMP-2 (Brunner et al., 2007; Hickey et al., 2009), identified a smaller subset of FLJ22263 proteins enriched in granules, as well as lysosomal and mitochondrial proteins likely due to co-sedimentation of these organelles during fractionation (Suckale and Solimena, 2010). Secretory granule membranes purified from mouse insulinoma MIN6 cells showed that p23, a p24 family protein involved in vesicular transport, was localized to the insulin secretory granule membrane (Hosaka et al., 2007). While these data provide a glimpse of the proteomic composition of islet cell types and the insulin secretory granule, they do not identify the proteins that interact directly or indirectly with insulin to manage its synthesis, folding, trafficking and packaging for storage in granules – the central axis dictating -cell function. Herein, we report an insulin biosynthetic interaction network (insulin BIN) that describes in molecular terms the secretory pathway of the islet -cell. Using a conformation based approach coupled to mass spectrometry, we characterized protein interactions for the immature insulin separately from interactions found with the mature insulin. To address the importance of highly abundant proteins recovered in the insulin BIN in -cell biology, we characterized one prominent insulin BIN protein, transmembrane protein 24 (TMEM24) (also referred to as C2 domain containing protein 2 like (C2CD2L)). Molecular analysis of TMEM24 identify it as a pancreatic islet enriched protein that plays a key role in regulating glucose sensitive insulin release from the reserve pool of granules. Our results now provide a database that can now be used to dissect the biochemical and molecular mechanisms responsible for -cell secretory pathway function in insulin biogenesis and release. RESULTS Defining the Insulin Biosynthetic Interaction Network (insulin BIN) During granule maturation, the conformational precursor of insulin (proinsulin) generated by co-translational insertion into the endoplasmic reticulum (ER) is transported to the Golgi where it is proteolytically cleaved to yield the mature insulin. (Furuta et al., 1998; Zhu et al., 2002) Insulin is then stored in two pools of.