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)