Summary Transmembrane proteins must adopt proper membrane topology to perform their function. In mammalian cells, the topology of transmembrane proteins is determined by the direction through which transmembrane helices are inserted into ER during their translation on ER-associated ribosomes. It has been assumed that protein translocation across the ER membranes is a constitutive process so that transmembrane proteins must adopt a fixed topology. This assumption has been challenged by our recent observation that the direction through which transmembrane helices are inserted into the ER can be reversed under certain physiological conditions. We reported that ceramide inverted the topology of two polytopic transmembrane proteins, namely TM4SF20 and CCR5. Since this regulatory mechanism does not flip transmembrane proteins that have already been synthesized but inverts the topology of newly synthesized proteins by changing the direction through which transmembrane helices are translocated across membranes, we designated this process as Regulated Alternative Translocation (RAT). This project is initiated to further characterize RAT by delineating the mechanism of this topological regulation. We will begin by testing the hypothesis that TRAM2 is the ceramide sensor that interacts with the nascent transmembrane helices subjected to RAT. The approaches developed for this study can be generalized to identify ceramide interactome, a finding that might reveal more signaling reactions mediated by the sphingolipid. These approaches may also be applied unbiasedly to identify proteins interacting with the nascent transmembrane helices subjected to RAT, thereby providing more mechanistic insights into this novel translocation regulation. This project will also identify proteins subject to RAT by a novel proteome-wide approach capable of measuring topology of transmembrane proteins globally. Achieving this part of the project will not only reveal the breadth of RAT but also provide essential experimental data for proteome- wide assembly of topology of transmembrane proteins. Considering that only 10% of mammalian transmembrane proteins have their topology defined by experimental evidence, accomplishing this project should greatly improve our understanding of transmembrane proteins.