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The biogenesis of secretory as well as transmembrane proteins requires the activity of the universally conserved protein-conducting channel (PCC), the Sec61 complex (SecY complex in bacteria). (1) In eukaryotic cells the PCC is located in the membrane of the endoplasmic reticulum where it can bind to translating ribosomes for cotranslational protein transport. The Sec complex consists of three subunits (Sec61α, β and γ) and provides an aqueous environment for the translocation of hydrophilic peptides as well as a lateral opening in the Sec61α subunit that has been proposed to act as a gate for the membrane partitioning of hydrophobic domains (2). A plug helix and a so-called pore ring are believed to seal the PCC against ion flow and are proposed to rearrange for accommodation of translocating peptides (2,3). Several crystal and cryo-electron microscopy structures revealed different conformations of closed and partially open Sec61 and SecY complexes (2,4-8). However, in none of these samples has the translocation state been unambiguously defined biochemically. Here we present cryo-electron microscopy structures of ribosome-bound Sec61 complexes engaged in translocation or membrane insertion of nascent peptides. Our data show that a hydrophilic peptide can translocate through the Sec complex with an essentially closed lateral gate and an only slightly rearranged central channel. Membrane insertion of a hydrophobic domain seems to occur with the Sec complex opening the proposed lateral gate while rearranging the plug to maintain an ion permeability barrier. Taken together, we provide a structural model for the basic activities of the Sec61 complex as a protein-conducting channel., Ribosome-protein-conducting channel (PCC) complexes were formed using a well-established in vitro protein translocation system: a wheat germ translation extract combined with canine pancreatic endoplasmic reticulum membranes (9). We chose nascent [...]

In eukaryotes, most secretory and membrane proteins are targeted by an N‐terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein‐conducting channel (PCC). In the post‐translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre‐opened state. Here, we present a cryo‐EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N‐terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C‐terminus of Sec63. Together, we obtained a near‐complete and integrated model of the active Sec complex. SYNOPSIS: The overall architecture of the eukaryotic SecY/Sec61 channel for post‐translational protein translocation, and how it recognizes N‐terminal signal sequences in transported proteins, remain unclear. Here, structural analyses reveal the molecular architecture of the engaged yeast Sec complex, which binds the signal sequence in a unique open conformation. Cryo‐EM structures of the heptameric Sec complex reveal the conformational transition from the partially open apo‐state to the fully open signal sequence‐bound state.The signal sequence binds the lateral gate of the Sec61 complex, which is stabilized by the transmembrane helices of the Sec62 subunit.The X‐ray structure of the N‐terminal cytoplasmic domain of Sec62 reveals a novel fold that is coordinated by the phosphorylated C‐terminal peptide of the Sec63 subunit. [ABSTRACT FROM AUTHOR]

The different enzymes which isomerase's on a group of carbon skeleton mutases have important functions in catabolic processes in human liver and bacterial fermentations. It is indicated that the involvement of cob(II) alaminorganic radical partnership with 2-methylenegluterate mutase is similar to that in glutamate mutase and thus would be useful in conjugating high frequency and pulsed electron paramagnetic resonance techniques.

The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S, 3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer ε2 (ε, 53.5 kDa) and S, a monomer (σ, 14.8 kDa). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins. Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme B12. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B12 resulted in the formation of a fully active glutamate mutase heterotetramer (ε2σ2) containing one molecule of coenzyme B12. EPR spectra of recombinant glutamate mutase, now available in sufficiently large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)-glutamate. The EPR signals (gx,y ≈ 2.1, gz = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(II)alamin (gx,y = 2.25). The spin concentration was 34-50% of the concentration of the enzyme (ε2σ2) coenzyme complex. The competitive inhibitors (25, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134 -148% of the enzyme concentration. Even (S)-[2,3,3,4,4-²H5] glutamate induced a signal significantly different to that of (5)-glutamate with an intensity of only 1%. These data suggest an involvement of the Co(II)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the perdeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species. It is discussed that the paramagnetic species represents a radical pair composed of Co(II) and an organic radical. Overnight incubation of glutamate mutase and coenzyme with substrate or one of the competitive inhibitors resulted in an inactive enzyme and cob(II)alamin, which was identified by EPR spectroscopy (gx,y - 2.25). This cob(II)alamin appeared to be very similar to a cob(II)amide present in inactive glutamate mutase preparations from C. cochlearium. [ABSTRACT FROM AUTHOR]

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is highly conserved from yeast to humans. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila, and Caenorhabditis elegans. NAC was suggested to protect the nascent chain from inappropriate early interactions with cytosolic factors. Eukaryotic NAC is a heterodimer with two subunits sharing substantial homology with each other. All sequenced archaebacterial genomes exhibit only one gene homologous to the NAC subunits. Here we present the first archaebacterial NAC homolog. It forms a homodimer, and as eukaryotic NAC it is associated with ribosomes and contacts the emerging nascent chain on the ribosome. We present the first crystal structure of a NAC protein revealing two structural features: (i) a novel unique protein fold that mediates dimerization of the complex, and (ii) a ubiquitin-associated domain that suggests a yet unidentified role for NAC in the cellular protein quality control system via the ubiquitination pathway. Based on the presented structure we propose a model for the eukaryotic heterodimeric NAC domain. [ABSTRACT FROM AUTHOR]