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 sequence 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 folding of the newly formed Ub moiety, which precludes its translocation and makes possible its cleavage by UBPs. However, if a sufficiently long spacer is present between the signal sequence and Ub, then by the time the Ub polypeptide emerges from the ribosome, the latter is already docked at the transmembrane channel, allowing the translocation of both 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 aspects of targeting in protein translocation, for distinguishing directly between cotranslational and posttranslational translocation, and for comparing the strengths of different signal sequences. This method should also be applicable to non-ER translocation.
Johnsson
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N
Division of Biology, California Institute of Technology, Pasadena 91125.
Varshavsky
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A
D000595:Amino Acid Sequence; D001692:Biological Transport; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics; D007425:Intracellular Membranes / Q000378:metabolism*; D008954:Models, Biological; D015335:Molecular Probes; D008969:Molecular Sequence Data; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D018271:Signal Recognition Particle / Q000502:physiology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics; D043324:beta-Fructofuranosidase
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D015335:Molecular Probes; D021382:Protein Sorting Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; D018271:Signal Recognition Particle; D014452:Ubiquitins; D014759:Viral Envelope Proteins; D003513:Cycloheximide; D013762:Tetrahydrofolate Dehydrogenase; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase