BIS 104 lingappa c fall 08 001

BIS 104 lingappa c fall 08 001 - always passivein the...

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Unformatted text preview: always passivein the translocation process. Presumably ? due-to. features of chain folding, the choite of the passen- gger' can significantly influence the efficiency of every known step in the' process of translocation. Furthermore, _ at least some passenger domains fold in such a way that they cannot be terminated by a given signal sequence. Nevertheless, the simple experiments described above es- tablished that, in principle, a signal sequence would redi- " rect a cytoplasmic protein into thesecretory pathway. . Although experiments are usually carried out to an- river _a specific question, they often are most valuable for the subsequent issues they raise and the new ways of- loo'kingat' a problem that they foster. The simple experi- ment described above not only provided 'a test that many critics of the signal hypothesis found compelling, but also contributedvto the realization that protein-chuneras were a pewerful approach to the study-- of specific sequences in— voivedin protein trafficldng. Subsequent work an traffick- ing of protein chimeras has allowed the. study of protein sequences that target proteins to other intracellular organ- elles besidEs those of the secretory pathway (e.g.; chloro- plasts and mitochondria). Similarly, sequences that cause proteins to stop on their way aeross particular membranes is crucial step in the assembly of integral membranepro~ acids forming three subregions (Fig. 20-1851), At the N- tenninal end of the signal, from one to seven amino acids form a polar region that usually includes from one to three positively charged residues such as lysine. This positively charged segment may promote initial attachment of the signal to the negatively charged ER membrane surface. (Many phospholipid molecules are negatively charged at their polar ends; see p. 156.) Following the positively charged subregion is a hydrophobic care that includes from at least 6 to 12. or more amino acids. The amino acids in this region are capable of forming an alpha helix or beta strand long enough to span the hydrophobic interior of a mem— brane. The ability of the core to assume a hydrophobic membrane-spanning structure is considered an essential feature that promotes insertion of the signal in the membrane interior. Following this insertion, a hydro- philic pore may open in the membrane to allow passage of the remaining polypeptide chain. Following the core is the third region of the signal, including the point at which the signal is cleaved after insertion of a protein into the membrane. in most signals this region contains amino acids with small, uncharged side chains at the first and third positions upstream of the cleavage site, and often an amino acid with a bulky, polar or charged side group at the second position upstream of the cleavage site (Fig. 20-18b). These stnic- tural features presumably take on a configuration rec- ognized by the enzyme removing the signal. teins) have been identified through theme of chimeric proteins. Finally, protein chimeras; have shown that a__ sig—- -= nal sequence can be totated far from the amino terminus and that translocation across the ER can be uncoupled from translation (see, for example, Ref. 4). These sorts of ' experimental manipulations have contributed to decipher— ing the intricate mechanisms of protein targeting, trans- lociition,-and trafficking and remain a fruitful line" of : investigation today.’ Acknowledgments _ I thank R. Hegde for critical reading of the manuscript. References ‘ Blobel, G., and Dobbers'tein, B. J. Cell Biol; 67:552 (1975). ‘ Lingappa, V. R.; Chaidez, 1.; Yost, C. 5.; and Hedgpeth, 1. Pros. Nat'l Ami. Sci. USA 81:45!) (1984). ’ Simon, K.; Peters. E; and Lingappa, V. R. I. Cell Biol. 104: 1165 (1987). ‘ Perara, 2.; Rothman R. E; and Lingappa V. R. Science 252:348— 51 (I986). ' I 5 Chuck, 5.: L, and Lingappa, V. R. Cell 68:1—"13 (I992). The Signal Peptidase The activity of the signal peptidase in removing the signal is a requirement for successful transfer of most ER—directed proteins. It the signal peptidase is inhibited, transfer is usually incomplete. In particular, proteins that would normally pass entirely through the mem- brane, such as secreted proteins, become "stuck" in the membrane if the signal peptidase activity is faulty. Presence of the signal peptidase is readily detected in isolated ER membranes by its specific activity in clipping off the signal. Although the enzyme has not as yet been precisely located in ER membranes, it is thought to face the inner compartment of ER cistemae or, in view of its extreme hydrophobic nature, to be buried entirely in the membrane interior. The signal peptidase has been isolated and partially purified in the Blobel laboratory. Both the eukaryotic signal peptidase and its bac- terial equivalent, signal peptidase I (see Supplement 20-1), are universal in their activity and can recognize and correctly cleave N-terminal signals from any source, either eukaryotic or prokaryol’ic. Stop-Transfer Signals Once a protein is directed to the ER by an N—tenninal signal, it may either remain inserted in the membrane or pass through the membrane to enter the solution enclosed within the ER cistemae. Retention in the ER 842 CHAPTER 20 Protein Sorting, Distribution, Secretion, and Endocytosis ...
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