HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Harley, C. A. Articles by Tipper, D. J. Search for Related Content PubMed PubMed Citation Articles by Harley, C. A. Articles by Tipper, D. J. Social Bookmarking                      What's this?

Volume 271, Number 40, Issue of October 4, 1996 pp. 24625-24633
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

---

The Role of Charged Residues in Determining Transmembrane Protein Insertion Orientation in Yeast

(Received for publication, April 30, 1996)

From the Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

The first 79 residues of the yeast Ste2p G protein-coupled pheromone receptor, including the negatively charged N-terminal domain, the first transmembrane segment, and the following positively charged cytoplasmic loop, has been fused to a Kex2p-cleavable -lactamase reporter. Insertion orientation was determined by analysis of cell-associated and secreted -lactamase activities and independently corroborated by analysis of membrane association and glycosylation patterns. This fusion inserts with exclusively N terminus exofacial (N_exo) topology, serving as a model type III membrane protein. Orientation is unaffected by removal of all three positively charged residues in the cytoplasmic loop or by deletion of all but 12 residues from the N-terminal domain. The residual 2 N-terminal charge apparently provides a signal sufficient to determine N_exo topology. This is entirely consistent with the statistically derived rule in which the charge difference, (C-N), counted for the 15 immediately flanking residues, is the primary topology determinant. Mutations altering (C-N) to zero favors N_exo insertion by 3 to 1, whereas increasingly negative values cause increasing inversion of orientation. All results are consistent with the charge difference rule and indicate that whereas positive charges promote cytoplasmic retention, negative charges promote translocation.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				D. Li, S. Fournel-Gigleux, L. Barre, G. Mulliert, P. Netter, J. Magdalou, and M. Ouzzine Identification of Aspartic Acid and Histidine Residues Mediating the Reaction Mechanism and the Substrate Specificity of the Human UDP-glucuronosyltransferases 1A J. Biol. Chem., December 14, 2007; 282(50): 36514 - 36524. [Abstract] [Full Text] [PDF]

				E. Kvam and D. S. Goldfarb Structure and function of nucleus-vacuole junctions: outer-nuclear-membrane targeting of Nvj1p and a role in tryptophan uptake J. Cell Sci., September 1, 2006; 119(17): 3622 - 3633. [Abstract] [Full Text] [PDF]

				S. Sadtler, B. Laube, A. Lashub, A. Nicke, H. Betz, and G. Schmalzing A Basic Cluster Determines Topology of the Cytoplasmic M3-M4 Loop of the Glycine Receptor alpha 1 Subunit J. Biol. Chem., May 2, 2003; 278(19): 16782 - 16790. [Abstract] [Full Text] [PDF]

				R. Kochl, M. Alken, C. Rutz, G. Krause, A. Oksche, W. Rosenthal, and R. Schulein The Signal Peptide of the G Protein-coupled Human Endothelin B Receptor Is Necessary for Translocation of the N-terminal Tail across the Endoplasmic Reticulum Membrane J. Biol. Chem., May 3, 2002; 277(18): 16131 - 16138. [Abstract] [Full Text] [PDF]

				B. M. Abell, S. High, and M. M. Moloney Membrane Protein Topology of Oleosin Is Constrained by Its Long Hydrophobic Domain J. Biol. Chem., March 1, 2002; 277(10): 8602 - 8610. [Abstract] [Full Text] [PDF]

				S. Swift, P. J. Sheridan, L. Covic, and A. Kuliopulos PAR1 Thrombin Receptor-G Protein Interactions. SEPARATION OF BINDING AND COUPLING DETERMINANTS IN THE Galpha SUBUNIT J. Biol. Chem., January 28, 2000; 275(4): 2627 - 2635. [Abstract] [Full Text] [PDF]

				D. Falcone, H. Do, A. E. Johnson, and D. W. Andrews Negatively Charged Residues in the IgM Stop-Transfer Effector Sequence Regulate Transmembrane Polypeptide Integration J. Biol. Chem., November 19, 1999; 274(47): 33661 - 33670. [Abstract] [Full Text] [PDF]

				C. Rutz, W. Rosenthal, and R. Schulein A Single Negatively Charged Residue Affects the Orientation of a Membrane Protein in the Inner Membrane of Escherichia coli Only When It Is Located Adjacent to a Transmembrane Domain J. Biol. Chem., November 19, 1999; 274(47): 33757 - 33763. [Abstract] [Full Text] [PDF]

				A. Odermatt, P. Arnold, A. Stauffer, B. M. Frey, and F. J. Frey The N-terminal Anchor Sequences of 11beta -Hydroxysteroid Dehydrogenases Determine Their Orientation in the Endoplasmic Reticulum Membrane J. Biol. Chem., October 1, 1999; 274(40): 28762 - 28770. [Abstract] [Full Text] [PDF]

				M. Sato and M. Mueckler A Conserved Amino Acid Motif (R-X-G-R-R) in the Glut1 Glucose Transporter Is an Important Determinant of Membrane Topology J. Biol. Chem., August 27, 1999; 274(35): 24721 - 24725. [Abstract] [Full Text] [PDF]

				M. Sato, R. Hresko, and M. Mueckler Testing the Charge Difference Hypothesis for the Assembly of a Eucaryotic Multispanning Membrane Protein J. Biol. Chem., September 25, 1998; 273(39): 25203 - 25208. [Abstract] [Full Text] [PDF]

				C. A. Harley, J. A. Holt, R. Turner, and D. J. Tipper Transmembrane Protein Insertion Orientation in Yeast Depends on the Charge Difference across Transmembrane Segments, Their Total Hydrophobicity, and Its Distribution J. Biol. Chem., September 18, 1998; 273(38): 24963 - 24971. [Abstract] [Full Text] [PDF]

				N. Rodriguez-Cousino, F. E. Nargang, R. Baardman, W. Neupert, R. Lill, and D. A. Court An Import Signal in the Cytosolic Domain of the Neurospora Mitochondrial Outer Membrane Protein TOM22 J. Biol. Chem., May 8, 1998; 273(19): 11527 - 11532. [Abstract] [Full Text] [PDF]

				D. Bayle, D. Weeks, and G. Sachs Identification of Membrane Insertion Sequences of the Rabbit Gastric Cholecystokinin-A Receptor by in Vitro Translation J. Biol. Chem., August 8, 1997; 272(32): 19697 - 19707. [Abstract] [Full Text] [PDF]

				G. Gafvelin, M. Sakaguchi, H. Andersson, and G. von Heijne Topological Rules for Membrane Protein Assembly in Eukaryotic Cells J. Biol. Chem., March 7, 1997; 272(10): 6119 - 6127. [Abstract] [Full Text] [PDF]

				D. J. Tipper and C. A Harley Yeast Genes Controlling Responses to Topogenic Signals in a Model Transmembrane Protein Mol. Biol. Cell, April 1, 2002; 13(4): 1158 - 1174. [Abstract] [Full Text] [PDF]