UBIQUITIN-ASSISTED DISSECTION OF PROTEIN-TRANSPORT ACROSS MEMBRANES

We describe a new way to analyze targeting in protein translocation. A fusion in which ubiquitin (Ub) is positioned between a signal sequenc e and a reporter domain is cleaved by Ub-specific proteases (UBPs) in the cytosol unless the fusion can 'escape' into a compartment such as the endoplasmic reticulum (ER). The critical step involves rapid foldi ng of the newly formed Ub moiety, which precludes its translocation an d makes possible its cleavage by UBPs. However, if a sufficiently long spacer is present between the signal sequence and Ub, then by the tim e the Ub polypeptide emerges from the ribosome, the latter is already docked at the transmembrane channel, allowing the translocation of bot h the Ub and reporter domains of the fusion into the ER. We show that Ub fusions can be used as in vivo probes for kinetic and stochastic as pects of targeting in protein translocation, for distinguishing direct ly between cotranslational and posttranslational translocation, and fo r comparing the strengths of different signal sequences. This method s hould also be applicable to non-ER translocation.