Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins.

The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent post-translational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after tobacco etch virus protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences.

Asparagine-linked glycosylation is not directly coupled to protein translocation across the endoplasmic reticulum in Saccharomyces cerevisiae.

Mammalian cells express two oligosaccharyltransferase complexes, STT3A and STT3B, that have distinct roles in N-linked glycosylation. The STT3A complex interacts directly with the protein translocation channel to mediate glycosylation of proteins using an N-terminal-to-C-terminal scanning mechanism. N-linked glycosylation of proteins in budding yeast has been assumed to be a cotranslational reaction. We have compared glycosylation of several glycoproteins in yeast and mammalian cells. Prosaposin, a cysteine-rich protein that contains STT3A-dependent glycosylation sites, is poorly glycosylated in yeast cells and STT3A-deficient human cells. In contrast, a protein with extreme C-terminal glycosylation sites was efficiently glycosylated in yeast by a posttranslocational mechanism. Posttranslocational glycosylation was also observed for carboxypeptidase Y-derived reporter proteins that contain closely spaced acceptor sites. A comparison of two recent protein structures indicates that the yeast OST is unable to interact with the yeast heptameric Sec complex via an evolutionarily conserved interface due to occupation of the OST binding site by the Sec63 protein. The efficiency of glycosylation in yeast is not enhanced for proteins that are translocated by the Sec61 or Ssh1 translocation channels instead of the Sec complex. We conclude that N-linked glycosylation and protein translocation are not directly coupled in yeast cells.

Conserved motifs on the cytoplasmic face of the protein translocation channel are critical for the transition between resting and active conformations.

The Sec61 complex is the primary cotranslational protein translocation channel in yeast (Saccharomyces cerevisiae). The structural transition between the closed inactive conformation of the Sec61 complex and its open and active conformation is thought to be promoted by binding of the ribosome nascent-chain complex to the cytoplasmic surface of the Sec61 complex. Here, we have analyzed new yeast Sec61 mutants that selectively interfere with cotranslational translocation across the endoplasmic reticulum. We found that a single substitution at the junction between transmembrane segment TM7 and the L6/7 loop interferes with cotranslational translocation by uncoupling ribosome binding to the L6/7 loop from the separation of the lateral gate transmembrane spans. Substitutions replacing basic residues with acidic residues in the C-terminal tail of Sec61 had an unanticipated impact upon binding of ribosomes to the Sec61 complex. We found that similar charge-reversal mutations in the N-terminal tail and in cytoplasmic loop L2/3 did not alter ribosome binding but interfered with translocation channel gating. These findings indicated that these segments are important for the structural transition between the inactive and active conformations of the Sec61 complex. In summary our results have identified additional cytosolic segments of the Sec61 complex important for promoting the structural transition between the closed and open conformations of the complex. We conclude that positively charged residues in multiple cytosolic segments, as well as bulky hydrophobic residues in the L6/7-TM7 junction, are required for cotranslational translocation or integration of membrane proteins by the Sec61 complex.

Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum.

Protein synthesis, transport, and N-glycosylation are coupled at the mammalian endoplasmic reticulum by complex formation of a ribosome, the Sec61 protein-conducting channel, and oligosaccharyltransferase (OST). Here we used different cryo-electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit STT3A (staurosporine and temperature sensitive 3A) revealed how it is integrated into the OST and how STT3-paralog specificity for translocon-associated OST is achieved. The OST subunit DC2 was placed at the interface between Sec61 and STT3A, where it acts as a versatile module for recruitment of STT3A-containing OST to the ribosome-Sec61 complex. This detailed structural view on the molecular architecture of the cotranslational machinery for N-glycosylation provides the basis for a mechanistic understanding of glycoprotein biogenesis at the endoplasmic reticulum.

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins.

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.

Protein translocation across the rough endoplasmic reticulum.

The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration.

A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function.

A critical event in protein translocation across the endoplasmic reticulum is the structural transition between the closed and open conformations of Sec61, the eukaryotic translocation channel. Channel opening allows signal sequence insertion into a gap between the N- and C-terminal halves of Sec61. We have identified a gating motif that regulates the transition between the closed and open channel conformations. Polar amino acid substitutions in the gating motif cause a gain-of-function phenotype that permits translocation of precursors with marginally hydrophobic signal sequences. In contrast, hydrophobic substitutions at certain residues in the gating motif cause a protein translocation defect. We conclude that the gating motif establishes the hydrophobicity threshold for functional insertion of a signal sequence into the Sec61 complex, thereby allowing the wild-type translocation channel to discriminate between authentic signal sequences and the less hydrophobic amino acid segments in cytosolic proteins. Bioinformatic analysis indicates that the gating motif is conserved between eubacterial and archaebacterial SecY and eukaryotic Sec61.

Understanding integration of α-helical membrane proteins: the next steps.

Integration of a protein into the endoplasmic reticulum (ER) membrane occurs through a series of multistep reactions that include targeting of ribosome-nascent polypeptide complexes to the ER, attachment of the ribosome to the protein translocation channel, lateral partitioning of α-helical transmembrane spans into the lipid bilayer, and folding of the lumenal, cytosolic and membrane-embedded domains of the protein. However, the molecular mechanisms and kinetics of these steps are still not entirely clear. To obtain a better understanding of the mechanism of membrane protein integration, we propose that it will be important to utilize in vivo experiments to examine the kinetics of membrane protein integration and in vitro experiments to characterize interactions between nascent membrane proteins, protein translocation factors and molecular chaperones.

Translocation channel gating kinetics balances protein translocation efficiency with signal sequence recognition fidelity.

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7, and TM8 in Sec61p) to expose the signal sequence-binding site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in channel gating. The translocation defect caused by L7 mutations could be suppressed by the prl class of sec61 alleles, which reduce the fidelity of signal sequence recognition. The prl mutants are proposed to act by destabilizing the closed conformation of the translocation channel. Our results indicate that the equilibrium between the open and closed conformations of the protein translocation channel maintains a balance between translocation activity and signal sequence recognition fidelity.

Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome.

The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined subnanometer-resolution cryo-electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data, we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state, the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain, contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation.

Translocation of proteins through the Sec61 and SecYEG channels.

The Sec61 and SecYEG translocation channels mediate the selective transport of proteins across the endoplasmic reticulum and bacterial inner membrane, respectively. These channels are also responsible for the integration of membrane proteins. To accomplish these two critical events in protein expression, the transport channels undergo conformational changes to permit the export of lumenal domains and the integration of transmembrane spans. Novel insight into how these channels open during protein translocation has been provided by a combination of the analysis of new channel structures, biochemical characterization of translocation intermediates, molecular dynamics simulations, and in vivo and in vitro analysis of structure-based Sec61 and SecY mutants.

An interaction between the SRP receptor and the translocon is critical during cotranslational protein translocation.

The signal recognition particle (SRP)-dependent targeting pathway facilitates rapid, efficient delivery of the ribosome-nascent chain complex (RNC) to the protein translocation channel. We test whether the SRP receptor (SR) locates a vacant protein translocation channel by interacting with the yeast Sec61 and Ssh1 translocons. Surprisingly, the slow growth and cotranslational translocation defects caused by deletion of the transmembrane (TM) span of yeast SRbeta (SRbeta-DeltaTM) are exaggerated when the SSH1 gene is disrupted. Disruption of the SBH2 gene, which encodes the beta subunit of the Ssh1p complex, likewise causes a growth defect when combined with SRbeta-DeltaTM. Cotranslational translocation defects in the ssh1DeltaSRbeta-DeltaTM mutant are explained by slow and inefficient in vivo gating of translocons by RNCs. A critical function for translocation channel beta subunits in the SR-channel interaction is supported by the observation that simultaneous deletion of Sbh1p and Sbh2p causes a defect in the cotranslational targeting pathway that is similar to the translocation defect caused by deletion of either subunit of the SR.

The tail end of membrane insertion.

Many membrane proteins are inserted into cellular membranes via a carboxy-terminal tail-anchor segment, but the mechanism of insertion is poorly understood. In this issue of Cell, Stefanovic and Hegde (2007) report the identification and initial characterization of a soluble ATP-dependent receptor for the insertion of newly synthesized tail-anchored membrane proteins.

Identification of cytoplasmic residues of Sec61p involved in ribosome binding and cotranslational translocation.

The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in the translocation of nascent polypeptides that use the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome-binding activity, indicating that the L6 and L8 sec61 mutants affect different steps in the cotranslational translocation pathway.

Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum.

We have analyzed the interactions between the signal recognition particle (SRP), the SRP receptor (SR), and the ribosome using GTPase assays, biosensor experiments, and ribosome binding assays. Possible mechanisms that could contribute to an enhanced affinity between the SR and the SRP-ribosome nascent chain complex to promote protein translocation under physiological ionic strength conditions have been explored. Ribosomes or 60S large ribosomal subunits activate the GTPase cycle of SRP54 and SRalpha by providing a platform for assembly of the SRP-SR complex. Biosensor experiments revealed high-affinity, saturable binding of ribosomes or large ribosomal subunits to the SR. Remarkably, the SR has a 100-fold higher affinity for the ribosome than for SRP. Proteoliposomes that contain the SR bind nontranslating ribosomes with an affinity comparable to that shown by the Sec61 complex. An NH2-terminal 319-residue segment of SRalpha is necessary and sufficient for binding of SR to the ribosome. We propose that the ribosome-SR interaction accelerates targeting of the ribosome nascent chain complex to the RER, while the SRP-SR interaction is crucial for maintaining the fidelity of the targeting reaction.

Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties.

Oligosaccharyltransferase (OST) is an integral membrane protein that catalyzes N-linked glycosylation of nascent proteins in the lumen of the endoplasmic reticulum. Although the yeast OST is an octamer assembled from nonhomologous subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Wbp1p, Swp1p, and Stt3p), the composition of the vertebrate OST was less well defined. The roles of specific OST subunits remained enigmatic. Here we show that genomes of most multicellular eukaryotes encode two homologs of Stt3p and mammals express two homologs of Ost3p. The Stt3p and Ost3p homologs are assembled together with the previously described mammalian OST subunits (ribophorins I and II, OST48, and DAD1) into complexes that differ significantly in enzymatic activity. Tissue and cell type-specific differences in expression of the Stt3p homologs suggest that the enzymatic properties of oligosaccharyltransferase are regulated in eukaryotes to respond to alterations in glycoprotein flux through the secretory pathway and may contribute to tissue-specific glycan heterogeneity.

Effect of high carbohydrate and high protein diets on microsomal fatty acid composition, fluidity and delta 6 desaturation activity in kidney and lung

La administración dietas hiperglucídicas e hiperproteicas suministradas a ratas durante 3 días produce respectivamente una disminución y un aumento en el cociente araquidonato/linoleato en los lípidos totales de microsomas de pulmón, riñon e hígado. En el hígado y el riñon este efecto está correlacionado con un significativo descenso de la actividad de la delta6 desaturasa para el caso de la dieta hiperflucídica y con un aumento de la misma actividad enzimática en la dieta hiperproteica. La actividad de la delta6 desaturasa, medida a través de la conversión del ácido 1-14**C linoleico a ácido alfa-linolénico, no se detectó en los microsomas de pulmón debido probablemente a la poca capacidad de este tejido para producir el éster de CoA del sustrato usado, y a que el cociente 20:4/18:2 en este tejido fue similar al del hígado bajo las condiciones dietéticas analizadas. La anisotropía de fluorescencia (r) del definilhexatrieno mostró diferencias significativas entre los tres tejidos analizados, efecto que se correlacionó con sus respectivos cocientes colesterol/fosfolípidos. Ambos parámetros fueron inferiores en los microsomas hepáticos que en los de los otros tejidos y permanecieron sin modificarse bajo los diferentes regímenes estudiados. Los resultados indican que el efecto de las dietas hiperhidrocarbonada e hiperproteica sobre la delta6 desaturasa no conduce a alteraciones aparentes en las propiedades físicas de las membranas microsomales

Effect of high carbohydrate and high protein diets on microsomal fatty acid composition, fluidity and delta 6 desaturation activity in kidney and lung

La administración dietas hiperglucídicas e hiperproteicas suministradas a ratas durante 3 días produce respectivamente una disminución y un aumento en el cociente araquidonato/linoleato en los lípidos totales de microsomas de pulmón, riñon e hígado. En el hígado y el riñon este efecto está correlacionado con un significativo descenso de la actividad de la delta6 desaturasa para el caso de la dieta hiperflucídica y con un aumento de la misma actividad enzimática en la dieta hiperproteica. La actividad de la delta6 desaturasa, medida a través de la conversión del ácido 1-14**C linoleico a ácido alfa-linolénico, no se detectó en los microsomas de pulmón debido probablemente a la poca capacidad de este tejido para producir el éster de CoA del sustrato usado, y a que el cociente 20:4/18:2 en este tejido fue similar al del hígado bajo las condiciones dietéticas analizadas. La anisotropía de fluorescencia (r) del definilhexatrieno mostró diferencias significativas entre los tres tejidos analizados, efecto que se correlacionó con sus respectivos cocientes colesterol/fosfolípidos. Ambos parámetros fueron inferiores en los microsomas hepáticos que en los de los otros tejidos y permanecieron sin modificarse bajo los diferentes regímenes estudiados. Los resultados indican que el efecto de las dietas hiperhidrocarbonada e hiperproteica sobre la delta6 desaturasa no conduce a alteraciones aparentes en las propiedades físicas de las membranas microsomales (AU)

Effect of dietary columbinic acid on the fatty acid composition and physical membrane properties of different tissues of EFA- deficient rats

Se estudió el efecto del agregado de ácido columbínico (5 trans, 9 cis, 12 cis octadeca-trienoico) a una dieta libre de grasas sobre la composición de ácidos grasos de distintos tejidos de rata, y estos datos se correlacionaron con las propiedades físicas de dichos tejidos. La ausencia de lípidos en la dieta produjo cambios en la composición de ácidos grasos que son característicos de la deficiencia de ácidos grasos esenciales (AGE). Se observó un incremento significativo del porcentaje relativo de ácidos grasos monoenoicos acompañado de una disminución de los ácidos linoleico y araquidónico y un aumento del ácido eicosa-5,8,11-trienoico en los homogenatos de hígado riñon, pulmón y bazo. El ácido columbínico agregado a una dieta libre de grasas durante 24 ó 48 horas se incorporó en los distintos tejidos, elongándose parcialmente al ácido eicosa-7 trans 11 cis, 14 cis-trienoico, pero sin ser desaturado. El ácido columbínico modificó el perfil de composición de ácidos grasos de los lípidos en los distintos tejidos, de manera tal que su porcentaje de distribución fue similar al observado en los animales no deficientes en AGE, excepto por el descenso del ácido linoleico. La ausencia de lípidos en la dieta produjo un incremento en la anisotropía de fluorescencia determinada con excitación continua (rs) del 1,6-difenil-1,3,5-hexatrieno (DPH) en microsomas hepáticos, que se corrigió con la administración de ácido columbínico durante 24 hr. Se concluye que el ácido columbínico produjo un efecto favorable de corto alcance sobre las propiedades físicas de la membrana microsomal hepática (rs) atribuible a las modificaciones en la composición de ácidos grasos. El ácido columbínico, por lo tanto, induciría también un efecto favorable a corto plazo sobre la producción de eicosanos, pero no así a largo plazo