title: N-acetylation and phosphorylation of Sec complex subunits in the ER membrane.
abstract: BACKGROUND
  Covalent modifications of proteins provide a mechanism to control protein function. Here, we have investigated modifications of the heptameric Sec complex which is responsible for post-translational protein import into the endoplasmic reticulum (ER). It consists of the Sec61 complex (Sec61p, Sbh1p, Sss1p) which on its own mediates cotranslational protein import into the ER and the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p). Little is known about the biogenesis and regulation of individual Sec complex subunits.
  
  
  RESULTS
  We show that Sbh1p when it is part of the Sec61 complex is phosphorylated on T5 which is flanked by proline residues. The phosphorylation site is conserved in mammalian Sec61ß, but only partially in birds, and not in other vertebrates or unicellular eukaryotes, suggesting convergent evolution. Mutation of T5 to A did not affect the ability of mutant Sbh1p to complement the growth defect in a Δsbh1Δsbh2 strain, and did not result in a hypophosphorylated protein which shows that alternate sites can be used by the T5 kinase. A survey of yeast phosphoproteome data shows that Sbh1p can be phosphorylated on multiple sites which are organized in two patches, one at the N-terminus of its cytosolic domain, the other proximal to the transmembrane domain. Surprisingly, although N-acetylation has been shown to interfere with ER targeting, we found that both Sbh1p and Sec62p are cotranslationally N-acetylated by NatA, and N-acetyl-proteome data indicate that Sec61p is modified by the same enzyme. Mutation of the N-acetylation site, however, did not affect Sec62p function in posttranslational protein import into the ER. Disabling NatA resulted in growth retardation, but not in co- or posttranslational translocation defects or instability of Sec62p or Sbh1p.
  
  
  CONCLUSIONS
  We conclude that N-acetylation of transmembrane and tail-anchored proteins does not interfere with their ER-targeting, and that Sbh1p phosphorylation on T5, which is not present in Sbh2p, plays a non-essential role specific to the Sec61 complex.
authors.affiliation: Department of Clinical Biochemistry, University College London Hospital, London, UK.
authors.forename: Christina
authors.initials: C
authors.lastname: Soromani
authors.forename: Naiyan
authors.initials: N
authors.lastname: Zeng
authors.forename: Klaus
authors.initials: K
authors.lastname: Hollemeyer
authors.forename: Elmar
authors.initials: E
authors.lastname: Heinzle
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Klein
authors.forename: Thomas
authors.initials: T
authors.lastname: Tretter
authors.forename: Matthew N J
authors.initials: MN
authors.lastname: Seaman
authors.forename: Karin
authors.initials: K
authors.lastname: Römisch
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D000069816:SEC Translocation Channels; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D026901:Membrane Transport Proteins; C101189:SBH2 protein, S cerevisiae; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; C074213:SEC62 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C101188:Sbh1 protein, S cerevisiae; D033921:Vesicular Transport Proteins