The yeast cotranslational translocation pathway is responsible for the integration of most membrane proteins into the endoplasmic reticulum. Targeting of the ribosome-nascent chain (RNC) complex to the Sec61 protein translocation channel is mediated by the signal recognition particle (SRP) and the SRP receptor. Binding of the RNC to the Sec61 complex initiates a structural transition between the closed and open conformations of the Sec61 complex that permits signal sequence insertion and opening of the lumenal and lateral gates of the protein translocation channel. We are using ubiquitin translocation assay (UTA) reporters to monitor the in vivo kinetics of translocation channel gating in response to single-spanning and multi-spanning integral membrane protein substrates. Our experiments have revealed that transmembrane spans and lumenal domains of multi-spanning membrane proteins are exposed to the cytosol during membrane protein integration. Research support was provided by the National Institutes of Health (GM035687).