How does Sec63 affect the conformation of Sec61 in yeast?

The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the Sec61 channel subunit Sbh1 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent signal peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-depen dent substrates nearby the lateral gate

Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.The 54 kDa subunit of the signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and membrane proteins and it contributes to the targeting of these precursors to the membrane of the endoplasmic reticulum (ER). At the ER membrane, the binding of the signal recognition particle (SRP) to its receptor triggers the release of SRP54 from its bound signal sequence and the nascent polypeptide is transferred to the Sec61 translocon for insertion into, or translocation across, the ER membrane. In the current article, we have characterized the specificity of anti-SRP54 autoantibodies, which are highly characteristic of polymyositis patients, and investigated the effect of these autoantibodies on the SRP function in vitro. We found that the anti-SRP54 autoantibodies had a pronounced and specific inhibitory effect upon the translocation of the secretory protein preprolactin when analysed using a cell-free system. Our mapping studies showed that the anti-SRP54 autoantibodies bind to the amino-terminal SRP54 N-domain and to the central SRP54 G-domain, but do not bind to the carboxy-terminal M-domain that is known to bind ER signal sequences. Nevertheless, anti-SRP54 autoantibodies interfere with signal-sequence binding to SRP54, most probably by steric hindrance. When the effect of anti-SRP autoantibodies on protein targeting the ER membrane was further investigated, we found that the autoantibodies prevent the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit. This observation supports a model where the binding of the homologous GTPase domains of SRP54 and the alpha-subunit of the SRP receptor to each other regulates the release of ER signal sequences from the SRP54 M-domain

The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences

The signal recognition particle (SRP) binds to signal sequences when they emerge from a translating ribosome and targets the complex of ribosome, nascent chain and SRP to the membrane of the rough endoplasmic reticulum (rER) allowing the co-translational translocation of the nascent chain. By photo-crosslinking it has been shown that the signal sequence of preprolactin(PPL) only interacts with the methionine-rich (NI) domain of the 54 kDa protein subunit (SRP54) of SRP. Here we show that (i) a signal-anchor sequence is likewise crosslinked only to the methionine-rich domain of SRP54,(ii) free SRP54 can interact with signal sequences independently of the other components of SRP, (iii) its M domain suffices to perform this function, and (iv) an essentially intact M domain is required for signal sequence recognition. Alkylation of the N+G domain in intact SRP54 with N-ethyl maleimide (NE), but not after cleavage with V8 protease, prevents the binding of a signal sequence to the M domain. This suggests a proximity between the N+G and M domains of SRP54 and raises the possibility that the role of the N+G domain may be to regulate the binding and/or the release of signal sequences

Structure and biosynthesis of the signal-sequence receptor

The signal-sequence receptor (SSR) has previously been shown to be a component of the environment which nascent polypeptides meet on passage through the endoplasmic reticulum (ER) membrane. We report here on the primary structure of the SSR as deduced from cDNA clones and from direct protein sequencing. The glycoprotein is synthesized with a cleavable amino-terminal signal sequence and contains only one classical membrane-spanning segment. Its insertion into the ER membrane during biosynthesis depends on the function of the signal-recognition particle. SSR shows a remarkable charge distribution with the amino terminus being highly negatively charged, and the cytoplasmic carboxyl terminus positively charged. The SSR can be phosphorylated in its cytoplasmic tail both in intact cells and in a cell-free system, suggesting a regulation of its function. The localization of the protein in the ER membrane was confirmed by immunofluorescence microscopy

The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by the retention of polymers of neuroserpin as inclusions within the endoplasmic reticulum (ER) of neurons. We have developed monoclonal antibodies that detect polymerized neuroserpin and have used COS-7 cells, stably transfected PC12 cell lines and transgenic Drosophila melanogaster to characterize the cellular handling of all four mutant forms of neuroserpin that cause FENIB. We show a direct correlation between the severity of the disease-causing mutation and the accumulation of neuroserpin polymers in cell and fly models of the disease. Moreover, mutant neuroserpin causes locomotor deficits in the fly allowing us to demonstrate a direct link between polymer accumulation and neuronal toxicity

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

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.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are