Post-translational Insertion of a Small Tail-Anchored Protein into the Membrane of the Endoplasmic Reticulum
Proteins destined for membrane insertion can be targeted to and inserted into the endoplasmic reticulum (ER) either during synthesis (cotranslationally) or after their synthesis is completed (post-translationally). While the cotranslational pathway is well characterized, much less is known about a post-translational pathway for targeting and insertion of membrane proteins. In this study post-translational targeting and insertion of the small, tail-anchored ER membrane protein RAMP4op was analyzed. RAMP4op has been chosen as a model because of its small overall length, carboxy terminal location of the transmembrane (TM) domain and the presence of the short cytoplasmic segment. This allows investigation to be focused on the significance of the TM domain in processes of ER targeting and insertion. Membrane targeting and insertion of RAMP4op was analyzed in the rabbit reticulocytes lysate in vitro translation system supplemented with rough microsomal membranes (RM) post-translationally. Upon insertion into the membrane, RAMP4op becomes N-glycosylated. This allows clear discrimination between cytosolic and membrane inserted forms of the protein. In this assay system, RAMP4op can be efficiently targeted and inserted into RM using a post-translational pathway dependent on ATP hydrolysis. In the absence of membranes and after release from the ribosome, RAMP4op was detected in a defined soluble cytosolic complex. Cytosolic RAMP4op could be maintained in an insertionally competent state for at least one hour. Chemical crosslinking was used to search for and analyze potential interacting partners of RAMP4op. In the absence of membranes, a single cytosolic, non-ribosomal protein of 40 kDa (p40) was discovered in the proximity of RAMP4op. The interaction with p40 is established via the TM domain of RAMP4op. This interaction is hydrophobic in nature since cross-linking between RAMP4op and p40 is abolished in the presence of a non-ionic detergent. In the presence of RM RAMP4op could not be cross-linked to p40. Cross-linking between these two proteins was re-established upon removal of membranes. This suggests that the interaction between RAMP4op and p40 is part of the pathway for post-translational targeting of RAMP4op. Treatment of RM with trypsin resulted in significantly reduced efficiency of RAMP4op insertion. This shows that ER membrane proteins are required for the efficient post-translational insertion of RAMP4op. Treatment of RM with trypsin in lower concentrations, sufficient to inactivate SRP receptor, had no effect on the efficiency of RAMP4op post-translational insertion. Therefore, functional SRP receptor is not required for the ER insertion of RAMP4op. In context of these findings, cytosolic factors that are possible candidates for p40 or may be involved in an ATP hydrolysis-dependent step during RAMP4op post-translational targeting/insertion are discussed.
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