HAU16008-03-5 - GG E T T NN GAA C R O S STT H EM E E M B R...

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REVIEW Protein Translocation Across Biological Membranes William Wickner 1 * and Randy Schekman 2 * Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work. Emerging technologies have enabled progress in understanding protein translocation. In vitro protein synthesis led to the discovery that the mRNAs for secreted proteins are attached to membranes, whereas cytosolic proteins are made on free polysomes ( 1–3 ). The junction between endoplasmic reticulum (ER)–bound polysomes and the membrane is tight enough to exclude protease ( 4 ), and nascent chains are discharged directly into the lumen ( 5 ). Mouse myeloma mRNA was found to encode a poly- peptide 1.5 kD larger than mature immuno- globulin light chain ( 6 ). The larger form was postulated to be a precursor with an N-terminal extension that specifies secretion, a research direction that culminated in the signal hypoth- esis ( 7 ). B Signal sequences [ were deciphered for proteins that crossed a membrane, includ- ing the ER, mitochondria, chloroplast, and bacterial envelope. Armed with a signal sequence, mature domains merely had to be susceptible to unfolding in order to translocate ( 8 , 9 ). The signal sequences of each organelle have shared motifs of polarity and structure but no sequence conservation. For example, bac- terial or ER export signals are basic at the N terminus, followed by a stretch of 8 to 14 apolar residues and a short cleavage motif that is recognized by a dedicated peptidase. Bacte- rial sec (secretion) genes, encoding proteins that support translocation, were identified either as suppressors of signal sequence mutants ( 10 ) or as temperature-sensitive mutants that failed to initiate secretion at the nonpermissive temperature ( 11 ). The yeast Sec61p ( 12 ) is homologous to bacterial SecY, establishing that the membrane-embedded portion of these translocons is conserved. In vitro translocation reactions were developed by adding isolated organelles to protein synthe- sis extracts for ER ( 7 ), mitochondria ( 13 , 14 ), chloroplasts ( 15 ), and bacterial plasma mem- brane ( 16 , 17 ). These in vitro systems were the basis for discovering energy requirements, determining whether translocation needed chaperones or ongoing protein synthesis, and
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HAU16008-03-5 - GG E T T NN GAA C R O S STT H EM E E M B R...

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