Elisabet Mandon

Structure of momoneric yeast and mammalian sec61 complexes interacting with the translating ribosome

Elisabet Mandon — Fri, 10 Feb 2012 05:57:00 PST

Role of Sec61alpha in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel

Weiqun Song et al. — Fri, 10 Feb 2012 05:39:30 PST

Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR). Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex. We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis. In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked. Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54.

A monomeric protein in the Golgi membrane catalyzes both N-deacetylation and N-sulfation of heparan sulfate

Elisabet C. Mandon et al. — Fri, 10 Feb 2012 05:39:29 PST

Recent studies have shown that the rat liver heparan sulfate N-deacetylase/N-sulfotransferase is a glycoprotein encoded by a single polypeptide chain of 882 amino acids. Using radiation inactivation analyses, we have now determined that in rat liver Golgi vesicles the target size for the N-deacetylase is 88 +/- 14 kDa, whereas that of the N-sulfotransferase is 92 +/- 8 kDa. These results, together with previous biochemical and molecular cloning approaches, demonstrate that 1) in rat liver Golgi membranes there exists only on population of molecules expressing both activities, 2) the active protein in the Golgi membrane functions as a monomer, and 3) there is no evidence that a large independent protein acts as a regulator of either activity.

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

Elisabet C. Mandon et al. — Fri, 10 Feb 2012 05:39:28 PST

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.

A mutant yeast deficient in Golgi transport of uridine diphosphate N-acetylglucosamine

Claudia Abeijon et al. — Fri, 10 Feb 2012 05:39:27 PST

Mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they have terminal alpha1-->2-linked N-acetylglucosamine and lack mannose phosphate. In a previous study, Douglas and Ballou (Douglas, R. K., and Ballou, C. E. (1982) Biochemistry 21, 1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins. The mutant had normal levels of N-acetylglucosaminyltransferase activity, and the partially purified enzyme from wild-type and mutant cells had the same apparent size, heat stability, affinity for substrates, metal requirement, and subcellular location. No qualitative or quantitative differences were found between mutant and wild-type cells in endogenous mannan acceptors and pools of UDP-GlcNAc. Chitin was synthesized at similar rates in wild-type and mutant cells, and the latter did not have a soluble inhibitor of the N-acetylglucosaminyltransferase or a hexosaminidase that could remove N-acetylglucosamine from mannoproteins. Together, the above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a defect in compartmentation of substrates involved in the biosynthesis of mannoproteins. We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis. Golgi vesicles which were sealed and of the same membrane topographical orientation as in vivo were isolated from wild-type and mnn2-2 mutant cells and incubated with UDP-GlcNAc in an assay in vitro. The initial rate of transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature dependent, saturable with an apparent Km of 5.5 microM and a Vmax of 8.2 pmol/mg of protein/3 min. No transport of UDP-GlcNAc was detected into Golgi vesicles from mutant cells. However, Golgi vesicles from both cells translocated GDP-mannose at comparable velocities, indicating that the above transport defect is specific. In addition to the above defect in mannoproteins, mutant cells were also deficient in the biosynthesis of glucosamine containing lipids.

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

Ying Jiang et al. — Fri, 10 Feb 2012 05:39:26 PST

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.

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

Zhiliang Cheng et al. — Fri, 10 Feb 2012 05:39:25 PST

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.

Purification of the Golgi adenosine 3'-phosphate 5'-phosphosulfate transporter, a homodimer within the membrane

Elisabet C. Mandon et al. — Fri, 10 Feb 2012 05:39:24 PST

Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen of the Golgi apparatus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the sulfate donor in these reactions, must be transported from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus. We have identified and purified to apparent homogeneity the rat liver Golgi membrane PAPS transporter by a combination of conventional and affinity chromatography as well as photoaffinity radiolabeling with adenosine 3',5'-bisphosphate, a competitive inhibitor of PAPS transport. The transporter, a 75-kDa protein, was purified 70,000-fold over homogenate (6% yield) and transported PAPS into phosphatidylcholine liposomes selectively and in a saturable manner (apparent Km of 1.7 microM). Radiation target-inactivation analyses of the transport activity in rat liver Golgi vesicles, together with the above described biochemical approaches, demonstrate that the PAPS transporter within the Golgi membrane is a homodimer.

Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

Claudia Abeijon et al. — Fri, 10 Feb 2012 05:39:23 PST

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.