Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Jun 30;275(26):19620-7.
doi: 10.1074/jbc.M001333200.

Interaction of murine BiP/GRP78 with the DnaJ homologue MTJ1

M Chevalier  1 , H Rhee, E C Elguindi, S Y Blond
Affiliations

Interaction of murine BiP/GRP78 with the DnaJ homologue MTJ1

M Chevalier et al. J Biol Chem. .

Abstract

The activity of Hsp70 proteins is regulated by accessory proteins, among which the most studied are the members of the DnaJ-like protein family. BiP/GRP78 chaperones the translocation and maturation of secreted and membrane proteins in the endoplasmic reticulum. No DnaJ-like partner has been described so far to regulate the function of mammalian BiP/GRP78. We show here that murine BiP/GRP78 interacts with the lumenal J domain of the murine transmembrane protein MTJ1 (J-MTJ1). J-MTJ1 stimulates the ATPase activity of BiP/GRP78 at stoichiometric concentrations. The C-terminal tail of BiP/GRP78 is not required for the interaction with J-MTJ1, leaving the function of this portion of the molecule still unclear. Physical interactions between J-MTJ1 and BiP/GRP78 are stable and can be abolished by a single histidine --> glutamine substitution in the highly conserved HPD motif shared by all DnaJ-like proteins. The J-MTJ1 fragment, but not the mutant J-MTJ1:H89Q fragment, stimulates the ATPase activity of Escherichia coli DnaK, although at a higher concentration than its genuine partner DnaJ. Full-length DnaJ does not stimulate BiP over the range of concentrations investigated. These results indicate that the J domain of MTJ1 is sufficient for its interaction with BiP/GRP78 and cannot be substituted by E. coli DnaJ.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Analysis of MTJ1 amino acid sequence (Swiss Protein Data Base accession number Q61712)
The transmembrane domains (long dashed underline) were predicted by using the TMPRED program. The signal peptide (dotted line) and cleavage site (arrow) were predicted with the program SignalP v1.1 (85). The DnaJ-like domain (triple underline) and the SANT domains (bold plain underline) were predicted by using the SMART program (86). The second region that presents homology with the yeast Sec63p (small dashed underline) has been identified using the alignment program SIM. The C-terminal endoplasmic reticulum retrieval signal (thick dotted line) was identified with the program PSORT II.
Fig. 2
Fig. 2. Potential topology of murine MTJ1 and yeast Sec63p
A, two models can be proposed for MTJ1 topology (see “Results”). B, the topology proposed by Feldheim and coworkers for Sec63p (51) is consistent with recent data obtained for a human homologue (80).
Fig. 3
Fig. 3. Purification and characterization of the J domain of MTJ1
Top panel, 15% SDS-PAGE analysis of His6-J-MTJ1 before and after cleavage by thrombin. Lane 1, uncleaved His6-J-MTJ1; lane 2, cleaved J-MTJ1; lane 3, purified cleaved J-MTJ1 not retained by the metal-chelating resin. Bottom panel, far-UV CD spectra of J-MTJ1 wild-type (left panel) and J-MTJ1:H89Q mutant (right panel). The protein concentrations were 11 and 8 μm for the wild-type and the mutant, respectively, in 10 mm sodium phosphate, pH 7.0. The open circles, the closed circles, and the closed triangles represent the experimental data, the fitted data (as calculated by the JFIT method), and the difference between experimental and fitted data, respectively.
Fig. 4
Fig. 4. Concentration-dependent stimulation of BiP ATPase activity by J-MTJ1
A, stimulation of His10-BiP steady-state ATPase activity by increasing concentrations of wild-type J-MTJ1 and mutant J-MTJ1:H89Q. His10-BiP (0.2 μm) was incubated with increasing concentrations (0.03–10 μm) of wild-type J-MTJ1 (closed circles) or mutant J-MTJ1:H89Q (open circles) and assayed for ATPase activity as described under “Experimental Procedures.” B, double reciprocal plot of BiP ATPase activity stimulated by J-MTJ1.
Fig. 5
Fig. 5. Complex formation between BiP and J-MTJ1
Interaction of His6-BiP with J-MTJ1 (left panels) or J-MTJ1: H89Q (right panels) was assayed as described under “Experimental Procedures.” Top panels, Coomassie-stained 15% SDS-PAGE. Lane 1, His6-BiP; lane 2; cleaved J-MTJ1 (or, right panel, mutant J-MTJ1:H89Q); lane 3, His6-BiP with wild-type J-MTJ1 (or, right panel, mutant J-MTJ1:H89Q); lane 4, His6-BiP with wild-type (or, right panel, mutant J-MTJ1:H89Q) in the presence of 1 mm ATP; lane 5, His6-BiP with wild-type (or, right panel, mutant J-MTJ1:H89Q) in the presence of 1 mm ADP. Bottom panel, densitometry scanning of the SDS-PAGE. The y axis represents the average of three independent experiments.
Fig. 6
Fig. 6. Effect of J-MTJ1 and J-MTJ1:H89Q on DnaK ATPase activity
A, steady-state stimulation of DnaK activity by J-MTJ1 or J-MTJ1:H89Q. DnaK (0.3 μm) was incubated with increasing concentrations (0.03–10 μm) of wild-type J-MTJ1 (closed circles) or J-MTJ1:H89Q (open circles) and assayed for ATPase activity. B, double reciprocal plot of J-MTJ1 stimulated ATPase activity of DnaK.
Fig. 7
Fig. 7. Effect of DnaJ on DnaK and BiP ATPase activity
A, steady-state stimulation of DnaK and BiP activity by DnaJ. 0.3 μm DnaK (closed circles) or His10-BiP (open circles) was incubated with increasing concentrations of DnaJ and assayed for ATPase activity. B, double reciprocal plot of DnaJ-stimulated ATPase activity of DnaK.
See this image and copyright information in PMC

References

    1. Lievremont JP, Rizzuto R, Hendershot L, Meldolesi J. J Biol Chem. 1997;272:30873–30879. - PubMed
    1. Hamman BD, Hendershot LM, Johnson AE. Cell. 1998;92:747–758. - PubMed
    1. Brodsky JL, Goeckeler J, Schekman R. Proc Natl Acad Sci U S A. 1995;92:9643–9646. - PMC - PubMed
    1. Corsi AK, Schekman R. J Biol Chem. 1996;271:30299–30302. - PubMed
    1. Lyman SK, Schekman R. Cell. 1997;88:85–96. - PubMed

Publication types

MeSH terms

LinkOut - more resources