Abstract
BackgroundCovalent 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.ResultsWe 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.ConclusionsWe 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.
Highlights
Covalent modifications of proteins provide a mechanism to control protein function
We find that two cotranslationally ERtargeted transmembrane proteins, Sec61p and Sec62p, are N-acetylated on a NatA consensus sequence (Figure 6 and [25]), suggesting that the binding of NatA and signal recognition particle (SRP) to the nascent protein are limiting, not their binding to the ribosome
That N-acetylation can occur on two cotranslationally targeted transmembrane proteins, Sec61p and Sec62p, but not on cotranslationally targeted soluble proteins [5], suggests that in signalsequence bearing proteins binding of NatA and SRP to the nascent protein are limiting, not their binding to the ribosome
Summary
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 Sec complex (Sec61p, Sbh1p, Sss1p) which on its own mediates cotranslational protein import into the ER and the Sec complex (Sec63p, Sec62p, Sec71p, Sec72p). The role of N-acetylation, remains unclear for the 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 Sec complex responsible for cotranslational protein import into the ER [6]. Deletion of either SBH1 or SBH2 does not affect yeast viability, but deletion of both genes leads to temperaturesensitivity [7]
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