Reconstitution of Secretory Protein Translocation from Detergent-Solubilized Rough Microsomes

NICCHITTA, CHRISTOPHER; Migliaccio, Giovanni; BLOBEL, GÜNTER

doi:10.1016/b978-0-12-683755-1.50011-9

Cited by 5 publications

(6 citation statements)

References 24 publications

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“…2 – 5 indicate that membrane-bound early (TVG60-mer) intermediates can be distinguished from late (TVG80-, 129-mer) intermediates by their sensitivity to extraction with high salt, yet the topology of the signal sequence for the different intermediates is identical, and all intermediates are bound to the membrane in a protease-resistant environment. To test the hypothesis that the differences in early- and late-stage intermediates may occur through late-stage specific interactions of translocation intermediates with components of the ER membrane, the ribosome–membrane junction for the TVG60-, 80-, and 129-mer intermediates was investigated in native and reconstituted membranes ( Nicchitta and Blobel, 1990 ; Nicchitta et al, 1991 ; Görlich et al, 1992 a ). Previously we have reported that reconstituted membranes, which lack the complement of lumenal chaperone/protein-folding enzymes, efficiently translocate nascent chains up to the stage of signal peptide cleavage, yet display reduced activity for net transfer of the nascent chain into the vesicle lumen ( Nicchitta and Blobel, 1990 ).…”

Section: Resultsmentioning

confidence: 99%

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Regulation of the Ribosome–Membrane Junction at Early Stages of Presecretory Protein Translocation in the Mammalian Endoplasmic Reticulum

Nicchitta

Zheng

1997

The Journal of Cell Biology

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A series of fusion protein constructs were designed to investigate the contribution of secretory nascent chains to regulation of the ribosome–membrane junction in the mammalian endoplasmic reticulum. As a component of these studies, the membrane topology of the signal sequence was determined at stages of protein translocation immediately after targeting and before signal sequence cleavage. Truncated translation products were used to delimit the analysis to defined stages of translocation.In a study of secretory protein precursors, formation of a protease-resistant ribosome–membrane junction, currently thought to define the pathway of the translocating nascent chain, was observed to be precursor- and stage-dependent. Analysis of the binding of early intermediates indicated that the nascent chain was bound to the membrane independent of the ribosome, and that the binding was predominately electrostatic. The membrane topology of the signal sequence was determined as a function of the stage of translocation, and was found to be identical for all assayed intermediates. Unexpectedly, the hydrophobic core of the signal sequence was observed to be accessible to the cytosolic face of the membrane at stages of translocation immediately after targeting as well as stages before signal sequence cleavage. Removal of the ribosome from bound intermediates did not disrupt subsequent translocation, suggesting that the active state of the protein-conducting channel is maintained in the absence of the bound ribosome. A model describing a potential mode of regulation of the ribosome–membrane junction by the nascent chain is presented.

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Section: Resultsmentioning

confidence: 99%

“…Lumenal protein-depleted RM were reconstituted by the procedures described in Nicchitta and Blobel (1990) and Nicchitta et al (1991) .…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Ribosome–Membrane Junction at Early Stages of Presecretory Protein Translocation in the Mammalian Endoplasmic Reticulum

Nicchitta

Zheng

1997

The Journal of Cell Biology

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“…Reconstitution of protein subfractions into proteoliposomes was accomplished by the detergent extraction procedure (Nicchitta et al ., 1991b) . For this procedure, SM 2 BioBeads (Bio-Rad Laboratories, Richmond, CA) were sequentially washed in methanol, water, and solubilization buffer.…”

Section: Reconstitution Ofvesiclesmentioning

confidence: 99%

The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

Migliaccio

Nicchitta

Blobel

1992

The Journal of Cell Biology

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Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.

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“…For deglycosylation, 10 p1 translation mix was incubated with 1000 U PNGase F (NEB) in 0.5% SDS, 1 % 2-mercaptoethanol, 1 % Nonidet P-40 and SO mM Na,HPO,, pH 7.5, for 1 h at 37°C. Translocation was monitored either at pH 7.5 in the presence of 20 mM EDTA, which dissociates the ribosomes and weakly bound proteins from the membranes [31], or the pH was adjusted to 11.5 with 100 mM NaOH, [32]. After 10-min incubation on ice, the neutral pH samples were overlayed on 300 p1 0.5 M sucrose, 50 mM triethanolamine, pH 7.5, 20 mM EDTA, while the NaOH-treated samples were overlayed on 300p1 0.2M sucrose, 60mM Hepes, pH 11.5, 50mM KOAc, 2.5 mM Mg(OAc), and 1 mM dithiothreitol.…”

Section: Glycosylation and Membrane Associationmentioning

confidence: 99%

Olfactory Receptor Proteins

Gat

Nekrasova

Lancet

et al. 1994

European Journal of Biochemistry

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A rat olfactory epithelium cDNA library was screened for olfactory receptor clones. One of the positively hybridizing cDNA clones was sequenced and found to encode a new member of the olfactory receptor superfamily. This cDNA, termed olp4, was used as a model of olfactory receptor for expression, both in vitro and in vivo. Expression of olp4, as well as of another previously cloned olfactory receptor (F5), was monitored by immunoprecipitation with a monoclonal antibody directed against a Flag peptide epitope tag, inserted at the N-terminus of the open reading frame, and a specific polyclonal antibody against a C-terminal peptide of olp4. Translation in v i m , followed by immunoprecipitation, showed a major olp4-specific band of 27-29 kDa. The olp4 and F5 polypeptides were found to be inserted into microsomal membranes as expected for integral membrane proteins. Expression in vivo of Flag-olp4 in Sf9 insect cells, using the baculovirus expression system, showed a specific polypeptide of the same size as the in vitro species, with an additional band of 34 kDa, which is most likely a glycosylated form. Fluorescence cytometry and immunohistochemical assays demonstrated the localization of the Flag-olp4 product on the cell surface of the infected host Sf9 cells, with the N-terminus and C-terminus in the proper orientation. Affinity chromatography was used for the partial purification of the olp4 polypeptide from infected Sf9 cells. The identification and purification of this expressed olfactory receptor polypeptide could open the way for further characterization and functional studies of the olfactory receptor superfamily members.The superfamily of olfactory receptor proteins [l, 21 is likely to underlie the detection of odorant ligands, thus constituting the molecular basis of the sense of smell. Olfactory receptors are seven-transmembrane-domain receptors, as suggested earlier on the basis of considerable evidence that odorant signals are transduced via GTP-binding proteins (Gproteins) on the cilia of olfactory sensory neurons to activate either the CAMP or the phosphoinositide cascades [3-71. Different olfactory receptors are believed to recognize different odorants, and odor coding is afforded by the specific expression of one or a few olfactory receptor types in each sensory neuron [ 8 -151.The first members of the olfactory receptor gene superfamily were cloned from rat and shown to encode several subfamilies [l]. Later, nearly 100 additional olfactory receptor genes were identified in several species [ l l , 16-19]. A systematic sequence classification by standard methods [20] shows a minimum of eight olfactory receptor families, each constituting one or more subfamilies, leading to the notion that the olfactory receptor gene repertoire is a multigene su- perfamily. Members of this superfamily share at least 35% amino acid sequence identity, as well as several structural features and sequence motifs that clearly distinguish them from the other seven-transmembrane-domain receptors [2]. Many of the olfactory re...

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Reconstitution of Secretory Protein Translocation from Detergent-Solubilized Rough Microsomes

Cited by 5 publications

References 24 publications

Regulation of the Ribosome–Membrane Junction at Early Stages of Presecretory Protein Translocation in the Mammalian Endoplasmic Reticulum

Regulation of the Ribosome–Membrane Junction at Early Stages of Presecretory Protein Translocation in the Mammalian Endoplasmic Reticulum

The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

Olfactory Receptor Proteins

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