Regulation of Protein Topology by trans-Acting Factors at the Endoplasmic Reticulum

Hegde, Ramanujan S.; Voigt, Sabine; Lingappa, Vishwanath R.

doi:10.1016/s1097-2765(00)80116-1

Cited by 91 publications

(100 citation statements)

References 20 publications

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“…2B (lanes 2, 5, and 8) shows that these mutations had no effect on PrP topology. As in the WT molecule, PrP containing either G20W or C22Y substitutions produced primarily Sec PrP, with ϳ10% being synthesized as Ctm PrP (Table I, lines [13][14][15]. This result was verified by assaying the topology of these mutants in transfected cells (data not shown).…”

Section: Lack Of Signal Peptide Cleavage Does Not Cause Production Ofmentioning

confidence: 72%

See 1 more Smart Citation

Mutational Analysis of Topological Determinants in Prion Protein (PrP) and Measurement of Transmembrane and Cytosolic PrP during Prion Infection

Stewart

Harris

2003

Journal of Biological Chemistry

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The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form ( Sec PrP), two transmembrane forms ( Ntm PrP and Ctm PrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, Ctm PrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of Ctm PrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of Ctm PrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the Ctm PrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases Ctm PrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes Ctm PrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of Ctm PrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that Ctm PrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.

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Section: Lack Of Signal Peptide Cleavage Does Not Cause Production Ofmentioning

confidence: 72%

“…The combined action of both domains operating during the transloca-tion process serves to regulate the proportions of the three topological variants of PrP. Regulatory factors associated with the translocon, in addition to sequence determinants within the PrP molecule itself, have also been shown to influence the final topology achieved (14,15).…”

mentioning

confidence: 99%

Mutational Analysis of Topological Determinants in Prion Protein (PrP) and Measurement of Transmembrane and Cytosolic PrP during Prion Infection

Stewart

Harris

2003

Journal of Biological Chemistry

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“…Intriguingly, the translocation of the prion protein that likewise has multiple folded states related to distinct functions with one particular form being responsible for neurodegeneration has been shown to be also a subject to regulation by as yet unidentified trans-acting cellular factors at the ER (4,5). Similarly, the topological variability of the P-glycoprotein, responsible for multidrug resistance observed in cancer cells, seems to be cotranslationally controlled by ribosomes (1,36).…”

Section: Discussionmentioning

confidence: 99%

“…However, certain proteins have been found to be expressed in two or more topological isoforms, with the heterogeneity apparently generated at the time of translocation at the endoplasmic reticulum (ER) membrane (1,2). In most of these cases, the topological diversity results in protein multifunctionality, suggesting regulatory mechanisms controlling translocational variations at the ER (3)(4)(5). One example of such a protein is the large L envelope protein of the hepatitis B virus (HBV), a polytopic membrane protein existing in a mixed topology (6)(7)(8).…”

mentioning

confidence: 99%

Chaperone action in the posttranslational topological reorientation of the hepatitis B virus large envelope protein: Implications for translocational regulation

Lambert

Prange

2003

Proc. Natl. Acad. Sci. U.S.A.

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The large L envelope protein of the hepatitis B virus utilizes a new folding pathway to acquire a dual transmembrane topology in the endoplasmic reticulum (ER). The process involves cotranslational membrane integration and subsequent posttranslational translocation of its preS subdomain into the ER. Here, we demonstrate that the conformational and functional heterogeneity of L depends on the action of molecular chaperones. Using coimmunoprecipitation, we observed specific interactions between L and the cytosolic Hsc70, in conjunction with Hsp40, and between L and the ERresident BiP in mammalian cells. Complex formation between L and Hsc70 was abolished when preS translocation was artificially switched to a cotranslational mode, implicating Hsc70 to act as a preS holding and folding catalyst that controls partial preS posttranslocation. The functional role of Hsc70 in L topogenesis was confirmed through modulation of its in vivo activity by overexpressing its co-chaperones Hip and Bag-1. Overexpression of the Hsc70-stimulating molecule Hip led to increased entrapping of preS on the cytosolic ER face and hence to a decrease in preS posttranslocation, whereas the negative regulator Bag-1 had the opposite effects. Furthermore, Hip-mediated Hsc70 activation impaired virus production in hepatitis B virus-replicating hepatoma cells, likely due to the improper topological reorientation of L. Together, these results indicate that translocational regulation of protein topology by chaperones provides a means of generating structural and functional diversity. They also hint to the dynamic nature of the mammalian ER translocation machinery in handling co-and posttranslational substrates.I t is generally thought that all copies of a given membrane protein exist in a single orientation with respect to the membrane. However, certain proteins have been found to be expressed in two or more topological isoforms, with the heterogeneity apparently generated at the time of translocation at the endoplasmic reticulum (ER) membrane (1, 2). In most of these cases, the topological diversity results in protein multifunctionality, suggesting regulatory mechanisms controlling translocational variations at the ER (3-5). One example of such a protein is the large L envelope protein of the hepatitis B virus (HBV), a polytopic membrane protein existing in a mixed topology (6-8).On biogenesis, the HBV L protein, together with the structurally closely related middle M and small S envelope proteins, is expressed from a single ORF of the viral genome by differential translation initiation. As a consequence, the entire sequence of S is repeated at the C termini of M and L, which contain the additional preS2 domain or preS2 and preS1 domains, respectively (9). All three proteins are cotranslationally integrated into the ER membrane by the topogenic signals of the S region that also direct cotranslational translocation of the upstream preS2 region of M into the ER lumen (10, 11). In contrast, the preS2 plus preS1 (preS) domain of L fails to be transloc...

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“…More recently, Hegde et al (1998b) have reported that the human prion protein can exist in several forms, including a secreted species and at least two topologically distinct integral membrane forms. The membrane insertion of PrP into reconstituted membranes requires the Sec61 complex and TRAM, but also requires unidentified ER components termed Translocation Accessory Factor( s) (TrAFs) (Hegde et al, 1998~). The extent to which these TrAFs might be generally involved in membrane protein assembly is currently unknown.…”

Section: Membrane-protein Insertionmentioning

confidence: 99%

Protein targeting to the endoplasmic reticulum in yeast

Stirling¹

1999

Microbiology

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Regulation of Protein Topology by trans-Acting Factors at the Endoplasmic Reticulum

Cited by 91 publications

References 20 publications

Mutational Analysis of Topological Determinants in Prion Protein (PrP) and Measurement of Transmembrane and Cytosolic PrP during Prion Infection

Mutational Analysis of Topological Determinants in Prion Protein (PrP) and Measurement of Transmembrane and Cytosolic PrP during Prion Infection

Chaperone action in the posttranslational topological reorientation of the hepatitis B virus large envelope protein: Implications for translocational regulation

Protein targeting to the endoplasmic reticulum in yeast

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