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Tom A. Rapoport - One of the best experts on this subject based on the ideXlab platform.

  • two alternative binding mechanisms connect the protein translocation sec71 sec72 complex with heat shock proteins
    Journal of Biological Chemistry, 2017
    Co-Authors: Arati Tripathi, Elisabet C Mandon, Reid Gilmore, Tom A. Rapoport
    Abstract:

    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.

  • targeting of rough Endoplasmic Reticulum Membrane proteins and ribosomes in invertebrate neurons
    Molecular Biology of the Cell, 2002
    Co-Authors: Melissa M Rolls, David H Hall, Martin Victor, Ernst H K Stelzer, Tom A. Rapoport
    Abstract:

    The Endoplasmic Reticulum (ER) is divided into rough and smooth domains (RER and SER). The two domains share most proteins, but RER is enriched in some Membrane proteins by an unknown mechanism. We studied RER protein targeting by expressing fluorescent protein fusions to ER Membrane proteins in Caenorhabditis elegans. In several cell types RER and general ER proteins colocalized, but in neurons RER proteins were concentrated in the cell body, whereas general ER proteins were also found in neurites. Surprisingly RER Membrane proteins diffused rapidly within the cell body, indicating they are not localized by immobilization. Ribosomes were also concentrated in the cell body, suggesting they may be in part responsible for targeting RER Membrane proteins.

  • a 12 residue long polyleucine tail is sufficient to anchor synaptobrevin to the Endoplasmic Reticulum Membrane
    Journal of Biological Chemistry, 1996
    Co-Authors: Paul Whitley, Ulrike Kutay, Tom A. Rapoport, Elin Grahn, Gunnar Von Heijne
    Abstract:

    Abstract Synaptobrevin is a tail-anchored protein with a hydrophobic C-terminal transMembrane segment that inserts into the Endoplasmic Reticulum Membrane independently of the SRP/Sec61p pathway. Here, we show that idealized hydrophobic segments composed of 11-17 leucines and 1 valine function as insertion signals in vitro, whereas shorter segments do not. These results suggest that there are no specific requirements beyond overall hydrophobicity for C-terminal Endoplasmic Reticulum insertion signals.

  • protein transport across the eukaryotic Endoplasmic Reticulum and bacterial inner Membranes
    Annual Review of Biochemistry, 1996
    Co-Authors: Tom A. Rapoport, Berit Jungnickel, Ulrike Kutay
    Abstract:

    Protein transport across the Endoplasmic Reticulum Membrane can occur by two pathways, a co- and a post-translational one. In both cases, polypeptides are first targeted to translocation sites in the Membrane by virtue of their signal sequences and then transported across or inserted into the phospholipid bilayer, most likely through a protein-conducting channel. Key components of the translocation apparatus have now been identified and the translocation pathways Seem likely to be related to each other but mechanistically distinct. Protein transport across the bacterial inner Membrane is both similar to and different from the process in eukaryotes. Other pathways of protein translocation exist that bypass the ones involving classical signal sequences.

  • a posttargeting signal sequence recognition event in the Endoplasmic Reticulum Membrane
    Cell, 1995
    Co-Authors: Berit Jungnickel, Tom A. Rapoport
    Abstract:

    Abstract We have analyzed early phases of the cotranslational transport of the secretory protein preprolactin through the mammalian Endoplasmic Reticulum (ER) Membrane. Following recognition of the signal sequence of the nascent polypeptide chain in the cytosol by the SRP, the chain is transferred into the Membrane, where a second signal sequence recognition step takes place for which the presence in the lipid bilayer of the Sec61 p complex is essential and sufficient. This step leads to a tight junction between the ribosomenascent chain complex and the Sec61p complex, and to the productive insertion of the nascent chain into the translocation site. These results show that a translocation substrate is subjected to two recognition events before being allowed to cross the ER Membrane.

Peter Walter - One of the best experts on this subject based on the ideXlab platform.

  • the unfolded protein response coordinates the production of Endoplasmic Reticulum protein and Endoplasmic Reticulum Membrane
    Molecular Biology of the Cell, 1997
    Co-Authors: Jeffery S Cox, Rowan Chapman, Peter Walter
    Abstract:

    The Endoplasmic Reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and Membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER Membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and Membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

  • signal sequences specify the targeting route to the Endoplasmic Reticulum Membrane
    Journal of Cell Biology, 1996
    Co-Authors: D T W Ng, Jeremy D Brown, Peter Walter
    Abstract:

    In the yeast Saccharomyces cerevisiae, only a subset of preproteins that are translocated across the ER Membrane require the function of the signal recognition particle (SRP), suggesting that an alternative, SRP-independent pathway must exist (Hann, B.C., and P. Walter. 1991. Cell. 67:131-144). We have established that the two targeting pathways function in parallel. Mutant alleles of SEC62 and SEC63 were isolated that specifically impaired the translocation of SRP-independent preproteins in vivo and in vitro, whereas SRP-dependent preproteins were unaffected. Based on this analysis, preproteins fall into three distinct classes: SRP dependent, SRP independent, and those that can use both pathways. Pathway specificity is conferred by the hydrophobic core of signal sequences. Our studies show a previously unrecognized diversity in ER-directed signal sequences, that carry structural information that serves to identify the route taken.

  • an amino terminal domain containing hydrophobic and hydrophilic sequences binds the signal recognition particle receptor α subunit to the β subunit on the Endoplasmic Reticulum Membrane
    Journal of Biological Chemistry, 1995
    Co-Authors: Jason C Young, Peter Walter, Josie Ursini, Kyle R Legate, Joshua D Miller, David W Andrews
    Abstract:

    The signal recognition particle receptor consists of two subunits of 72 kDa (SRgα) and 30 kDa (SRβ). Assembly of SRα on the Endoplasmic Reticulum Membrane can occur independent of the signal recognition particle-mediated translocation pathway. To identify the sequences within SRα necessary for Membrane binding, a series of amino-terminal and internal deletion mutants was constructed and translated in a cell-free system. In addition, nascent SRα polypeptides of varying lengths were generated by cycloheximide treatment of translation reactions. Microsome binding assays performed on these polypeptides revealed a Membrane binding domain consisting of the amino-terminal 140 residues of SRα. This domain includes the two hydrophobic sequences originally proposed to bind to Membranes and a highly charged region not previously implicated in Membrane assembly. Furthermore, the domain forms a protease-resistant folding unit that after proteolysis can target and anchor onto microsomes. Extraction of microsomal SRα at high pH supplemented with 1 M NaSCN suggests that SRα and the Membrane binding domain are not integrated in the Endoplasmic Reticulum Membrane. The Membrane binding domain is also the major site of tight binding with SRβ, suggesting that SRβ plays a role in the Membrane assembly of SRα.

  • interaction of e coli ffh 4 5s ribonucleoprotein and ftsy mimics that of mammalian signal recognition particle and its receptor
    Nature, 1994
    Co-Authors: Joshua D Miller, Harris D Bernstein, Peter Walter
    Abstract:

    The mechanism of protein translocation across the Endoplasmic Reticulum Membrane of eukaryotic cells and the plasma Membrane of prokaryotic cells are thought to be evolutionarily related. Protein targeting to the eukaryotic translocation apparatus is mediated by the signal recognition particle (SRP), a cytosolic ribonucleoprotein, and the SRP receptor, an Endoplasmic Reticulum Membrane protein. During targeting, the 54K SRP subunit (M(r) 54,000; SRP54), a GTP-binding protein, binds to signal sequences and then interacts with the alpha-subunit of the SRP receptor (SR alpha), another GTP-binding protein. Two proteins from Escherichia coli, Ffh and FTsY, structurally resemble SRP54 and SR alpha. Like SRP54, Ffh is a subunit of a cytosolic ribonucleoprotein that also contains the E. coli 4.5S RNA. Although there is genetic and biochemical evidence that the E. coli Ffh/4.5S ribonucleoprotein has an SRP-like function, there is no evidence for an SR alpha-like role for FtsY. Here we show that the Ffh/4.5S ribonucleoprotein binds tightly to FtsY in a GTP-dependent manner. This interaction results in the stimulation of GTP hydrolysis which can be inhibited by synthetic signal peptides. These properties mimic those of mammalian SRP and its receptor, suggesting that the E. coli Ffh/4.5S ribonucleoprotein and FtsY have functions in protein targeting that are similar to those of their mammalian counterparts.

  • signal sequence recognition and protein targeting to the Endoplasmic Reticulum Membrane
    Annual Review of Cell Biology, 1994
    Co-Authors: Peter Walter, Arthur E Johnson
    Abstract:

    STRUCTURE OF THE SRP AND SRP RECEPTOR . NOlllelleiatllre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SRP RNA SRP Structllre alld Assembly . SRP Proteill SlIbllllits . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SRP Receptor .

Youngsoo Jun - One of the best experts on this subject based on the ideXlab platform.

Ulrike Kutay - One of the best experts on this subject based on the ideXlab platform.

  • a 12 residue long polyleucine tail is sufficient to anchor synaptobrevin to the Endoplasmic Reticulum Membrane
    Journal of Biological Chemistry, 1996
    Co-Authors: Paul Whitley, Ulrike Kutay, Tom A. Rapoport, Elin Grahn, Gunnar Von Heijne
    Abstract:

    Abstract Synaptobrevin is a tail-anchored protein with a hydrophobic C-terminal transMembrane segment that inserts into the Endoplasmic Reticulum Membrane independently of the SRP/Sec61p pathway. Here, we show that idealized hydrophobic segments composed of 11-17 leucines and 1 valine function as insertion signals in vitro, whereas shorter segments do not. These results suggest that there are no specific requirements beyond overall hydrophobicity for C-terminal Endoplasmic Reticulum insertion signals.

  • protein transport across the eukaryotic Endoplasmic Reticulum and bacterial inner Membranes
    Annual Review of Biochemistry, 1996
    Co-Authors: Tom A. Rapoport, Berit Jungnickel, Ulrike Kutay
    Abstract:

    Protein transport across the Endoplasmic Reticulum Membrane can occur by two pathways, a co- and a post-translational one. In both cases, polypeptides are first targeted to translocation sites in the Membrane by virtue of their signal sequences and then transported across or inserted into the phospholipid bilayer, most likely through a protein-conducting channel. Key components of the translocation apparatus have now been identified and the translocation pathways Seem likely to be related to each other but mechanistically distinct. Protein transport across the bacterial inner Membrane is both similar to and different from the process in eukaryotes. Other pathways of protein translocation exist that bypass the ones involving classical signal sequences.

  • Transport route for synaptobrevin via a novel pathway of insertion into the Endoplasmic Reticulum Membrane.
    The EMBO journal, 1995
    Co-Authors: Ulrike Kutay, Gudrun Ahnert-hilger, Enno Hartmann, B Wiedenmann, Tom A. Rapoport
    Abstract:

    Synaptobrevin/vesicle-associated Membrane protein is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is proposed to provide specificity for the targeting and fusion of vesicles with the plasma Membrane. It belongs to a class of Membrane proteins which lack a signal sequence and contain a single hydrophobic segment close to their C-terminus, leaving most of the polypeptide chain in the cytoplasm (tail-anchored). We show that in neuroendocrine PC12 cells, synaptobrevin is not directly incorporated into the target organelle, synaptic-like vesicles. Rather, it is first inserted into the Endoplasmic Reticulum (ER) Membrane and is then transported via the Golgi apparatus. Its insertion into the ER Membrane in vitro occurs post-translationally, is dependent on ATP and results in a trans-Membrane orientation of the hydrophobic tail. Membrane integration requires ER protein(s) different from the translocation components needed for proteins with signal sequences, thus suggesting a novel mechanism of insertion.

Changwook Lee - One of the best experts on this subject based on the ideXlab platform.