The membrane-interactive tail of cytochrome b5 can function as a stop-transfer sequence in concert with a signal sequence to give inversion of protein topology in the endoplasmic reticulum

Human CYP17 (P-45017α, 17α-hydroxylase-17,20-lyase)-catalysed side-chain cleavage of 17α-hydroxyprogestogens into androgens is greatly dependent on the presence of cytochrome b5. The native form of cytochrome b5 is composed of a globular core, residues 1Ő98, followed by a membrane insertable C-terminal tail, residues 99Ő133. In the present study the abilities of five different forms of cytochrome b5 to support the side-chain cleavage activity of CYP17 were compared. The five derivatives were: the native pig cytochrome b5 (native pig), its genetically engineered rat counterpart (coreŐtail), the soluble core form of the latter (core), the core with the secretory signal sequence of alkaline phosphatase appended to its N-terminal (signalŐcore) and the latter containing the C-terminal tail of the native rat protein (signalŐcoreŐtail). When examined by Edman degradation and MS, the engineered proteins were shown to have the expected N-terminal amino acid sequences and molecular masses. The native pig was found to be acetylated at the N-terminal. The native pig and coreŐtail enzymes were equally efficient at enhancing the side-chain cleavage activity of human CYP17 and the signalŐcoreŐtail was 55% as efficient. The core and signalŐcore constructs were completely inactive in the aforementioned reaction. All the five derivatives were reduced to varying degrees by NADPH:cytochrome P-450 (NADPH-P450) reductase and the relative efficiencies of this reduction were reminiscent of the behaviour of these derivatives in supporting the side-chain cleavage reaction. In the side-chain cleavage assay, however, NADPH-P450 reductase was used in large excess so that the reduction of cytochrome b5 derivatives was not rate-limiting. The results highlight that productive interaction between cytochrome b5 and CYP17 is governed not only by the presence of a membrane insertable hydrophobic region on the cytochrome b5 but also by its defined spatial orientation at the C-terminal.

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Efficiency and diversity of protein localization by random signal sequences

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3163-3173.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 3163-3173

Author(s):

C A Kaiser ◽

D Botstein

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Signal Peptide ◽

Signal Sequence ◽

Protein Localization ◽

Random Signal ◽

Perinuclear Region ◽

Hybrid Protein ◽

Random Sequences ◽

Beta Galactosidase

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

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Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation

Journal of Virology ◽

10.1128/jvi.01700-08 ◽

2009 ◽

Vol 83 (19) ◽

pp. 10075-10084 ◽

Cited By ~ 7

Author(s):

Alenka Jejcic ◽

Robert Daniels ◽

Laura Goobar-Larsson ◽

Daniel N. Hebert ◽

Anders Vahlne

Keyword(s):

Human Immunodeficiency Virus ◽

Endoplasmic Reticulum ◽

Protein Degradation ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Signal Sequence ◽

Immunodeficiency Virus ◽

Steady State Levels ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH2), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH2 was uncovered by examining Env expression and maturation in treated cells. GPG-NH2 treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH2 negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH2, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.

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Signal Sequence Recognition and Protein Targeting to the Endoplasmic Reticulum Membrane

Annual Review of Cell Biology ◽

10.1146/annurev.cb.10.110194.000511 ◽

1994 ◽

Vol 10 (1) ◽

pp. 87-119 ◽

Cited By ~ 479

Author(s):

Peter Walter ◽

Arthur E. Johnson

Keyword(s):

Endoplasmic Reticulum ◽

Protein Targeting ◽

Signal Sequence ◽

Endoplasmic Reticulum Membrane ◽

Sequence Recognition

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Chapter 13 Analysis of Protein Topology in the Endoplasmic Reticulum

Methods in Cell Biology - Vectorial Pansport of Proteins into and across Membranes ◽

10.1016/s0091-679x(08)61686-6 ◽

1991 ◽

pp. 287-302 ◽

Cited By ~ 19

Author(s):

Hans Peter Wessels ◽

James P. Beltzer ◽

Martin Spiess

Keyword(s):

Endoplasmic Reticulum ◽

Protein Topology

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HiPER1, a phosphatase of the endoplasmic reticulum with a role in chondrocyte maturation

Journal of Cell Science ◽

10.1242/jcs.111.6.803 ◽

1998 ◽

Vol 111 (6) ◽

pp. 803-813

Author(s):

P.R. Romano ◽

J. Wang ◽

R.J. O'Keefe ◽

J.E. Puzas ◽

R.N. Rosier ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Growth Plate ◽

Articular Chondrocytes ◽

Signal Sequence ◽

Growth Plate Chondrocytes ◽

Chondrocyte Maturation ◽

Er Retention ◽

Alternatively Spliced ◽

Dual Label

We have previously identified and partially cloned Band 17, a gene expressed in growth plate chondrocytes transiting from proliferation to hypertrophy. We now rename this gene HiPER1, Histidine Phosphatase of the Endoplasmic Reticulum-1, based on the results reported here. HiPER1 encodes two proteins of 318 (HiPER1(318)) and 449 (HiPER1(449)) amino acids, which are 20–21% identical to a group of yeast acid phosphatases that are in the histidine phosphatase family. HiPER1(449) is significantly more abundant than HiPER1(318), correlating with the abundance of the alternatively spliced messages encoding HiPER449 and HiPER318. Anti-HiPER1 antibodies detect two proteins of 53 and 55 kDa in growth plate chondrocytes that are absent in articular chondrocytes. We confirm that the 53 and 55 kDa proteins are HiPER1(449) by heterologous expression of the HiPER1(449) coding sequence in chick embryo fibroblasts. The 53 and 55 kDa proteins are glycosylated forms of HiPER1(449), as N-glycosidase F digestion reduces these proteins to 48 kDa, the predicted size of HiPER1(449) without the N-terminal signal sequence. Immunocytochemistry demonstrates that HiPER1(449) is found in chondrocytes maturing from proliferation to hypertrophy, but is not detectable in resting zone, deep hypertrophic zone or articular chondrocytes, a distribution that is consistent with the message distribution. HiPER1(449) was predicted to localize to the lumen of endoplasmic reticulum by an N-terminal signal sequence and by the C-terminal sequence Ala-Asp-Glu-Leu, which closely matches the consensus signal for ER retention, Lys-Asp-Glu-Leu. We confirm this prediction by demonstrating colocalization of HiPER1(449) with the ER protein HSP47 using dual-label immunofluorescence. PTHrP, a peptide that prevents hypertrophy in chondrocytes, suppressed HiPER1 and HiPER1(449) expression in vitro, an observation that further supports a role for HiPER1 in chondrocyte maturation. The yeast phosphatase homology, localization to the endoplasmic reticulum and pattern of expression suggest that HiPER1 represents a previously unrecognized intracellular pathway, involved in differentiation of chondrocytes.

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