N-acetylation and phosphorylation of Sec complex subunits in the ER membrane

BMC Cell Biology, Dec 2012

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.

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N-acetylation and phosphorylation of Sec complex subunits in the ER membrane

BMC Cell Biology N-acetylation and phosphorylation of Sec complex subunits in the ER membrane Christina Soromani 3 Naiyan Zeng 4 Klaus Hollemeyer 1 Elmar Heinzle 1 Marie-Christine Klein 0 Thomas Tretter 0 Matthew N J Seaman 2 Karin Römisch 0 0 Department of Microbiology, Faculty of Biology, Saarland University , Campus A1.5, 66123 Saarbruecken , Germany 1 Department of Technical Biochemistry, Faculty of Chemistry, Saarland University , Saarland , Germany 2 CIMR , Cambridge , UK 3 Department of Clinical Biochemistry, University College London Hospital , London , UK 4 Shanghai Jiao-Tong University School of Medicine , Shanghai , China 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. Protein translocation; Endoplasmic Reticulum; Sec complex; Sbh1p; Sec62p; Sec61p; Phosphorylation; N-acetylation; Convergent evolution; ER targeting Background Protein phosphorylation is a reversible mechanism used in all kingdoms of life to regulate protein activity, location and stability [ 1 ]. Protein N-acetylation which is irreversible can regulate protein stability and protein-protein interactions [ 2,3 ]. Many proteins (50% in yeast) are N-acetylated, the enzymes responsible for N-acetylation have been identified, and their substrate specificities characterized [4]. The role of N-acetylation, however, remains unclear for the majority of substrates to date. Strikingly, proteins bearing N-terminal signal sequences are usually not N-acetylated [ 5 ]. If the modification is introduced by mutation, Nacetylation leads to missorting of secretory proteins to the cytosol. These observations led to the conclusion that Nacetylation interferes with ER targeting [ 5 ]. Although protein flux across the ER membrane can be extremely variable, nothing is known about the regulation of the activity of the protein translocation channel in the ER membrane. In yeast the channel is composed of 3 subunits, Sec61p, Sbh1p and Sss1p, which form the Sec61 complex responsible for cotranslational protein import into the ER [ 6 ]. The channel subunits are highly conserved with mammalian proteins Sec61α, Sec61ß and Sec61γ. In yeast, posttranslational import into the ER of proteins with less hydrophobic signal sequences is mediated by a heptameric complex which in addition to the Sec61 complex contains the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p) [ 6 ]. Yeast also express a homologue of Sec61p, Ssh1p, which together with Sss1p and a homologue of Sbh1p, Sbh2p, forms the Ssh1 complex responsible exclusively for co-translational import into the ER [ 6 ]. Protein translocation into the ER and the SEC61, SSS1, SEC63 and SEC62 genes are essential. Deletion of either SBH1 or SBH2 does not affect yeast viability, but deletion of both genes leads to temperaturesensitivity [ 7 ]. Sbh1p and Sbh2p interact with multiple partners, and it is not known how these interactions are regulated. Sbh1p and Sbh2p are small tail-anchored proteins in the ER membrane with large (...truncated)


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Christina Soromani, Naiyan Zeng, Klaus Hollemeyer, Elmar Heinzle, Marie-Christine Klein, Thomas Tretter, Matthew N J Seaman, Karin Römisch. N-acetylation and phosphorylation of Sec complex subunits in the ER membrane, BMC Cell Biology, 2012, pp. 34, 13, DOI: 10.1186/1471-2121-13-34