title: The positive inside rule is stronger when followed by a transmembrane helix.
abstract: The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.
authors.affiliation: Science for Life Laboratory, Stockholm University, SE-171 21 Solna, Sweden; Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address: minttu.virkki@scilifelab.se.
authors.forename: Minttu T
authors.initials: MT
authors.lastname: Virkki
authors.affiliation: Science for Life Laboratory, Stockholm University, SE-171 21 Solna, Sweden; Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; Swedish e-Science Research Center (SeRC), SE-100 44 Stockholm, Sweden. Electronic address: christoph.peters@scilifelab.se.
authors.forename: Christoph
authors.initials: C
authors.lastname: Peters
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address: daniel.nilsson@dbb.su.se.
authors.forename: Daniel
authors.initials: D
authors.lastname: Nilsson
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address: therese@cormentia.se.
authors.forename: Therese
authors.initials: T
authors.lastname: Sörensen
authors.affiliation: Department of Clinical and Experimental Medicine, Cell Biology, Faculty of Health Science, Linköping University, 581 83 Linköping, Sweden; Department of Physiology, IKERBASQUE, Basque Foundation for Science, Faculty of Medicine and Dentistry, University of the Basque Country, 48949 Leioa, Spain. Electronic address: susana.cristobal@liu.se.
authors.forename: Susana
authors.initials: S
authors.lastname: Cristobal
authors.affiliation: Swedish e-Science Research Center (SeRC), SE-100 44 Stockholm, Sweden; Department of Physics, Chemistry and Biology, Linköping University, 581 83 Linköping, Sweden. Electronic address: bjorn.wallner@liu.se.
authors.forename: Björn
authors.initials: B
authors.lastname: Wallner
authors.affiliation: Science for Life Laboratory, Stockholm University, SE-171 21 Solna, Sweden; Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; Swedish e-Science Research Center (SeRC), SE-100 44 Stockholm, Sweden. Electronic address: arne@bioinfo.se.
authors.forename: Arne
authors.initials: A
authors.lastname: Elofsson
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D004285:Dogs; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010179:Pancreas / Q000378:metabolism; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D001120:Arginine; D012697:Serine Endopeptidases; C026515:type I signal peptidase
keywords: marginally hydrophobic helices; membrane proteins; orientational preference; positive inside rule; translocon recognition