doi: 10.1093/emboj/cdf315.
A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes
Affiliations
- PMID: 12065409
- PMCID: PMC126068
- DOI: 10.1093/emboj/cdf315
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A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes
EMBO J.
.
Erratum in
- EMBO J 2002 Jul 15;21(14):3917
Abstract
Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ-like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK-like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ-like membrane protein, which had been termed Mtj1p in mouse. Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP. In addition to a lumenal J-domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation. The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p. We propose that Mtj1p represents a novel type of co-chaperone, mediating transmembrane recruitment of DnaK-like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK-like chaperones of the endoplasmic reticulum.
Figures
Fig. 1. Sequence of Mtj1p. Sequence of murine Mtj1p (Brightman et al., 1995) and the N-terminal sequence of canine Mtj1p (shown below the murine sequence). The protein sequence data for canine Mtj1p will appear in the SWISS-PROT Protein Data Bank under accession number P82539. Note that: (i) two dots represent an identical amino acid residue, present in the canine and the murine protein; (ii) x represents an ambiguous result in the amino acid analysis; (iii) the underlined murine sequences represent putative transmembrane domains; (iv) italics indicate the J-domain; and (v) peptide sequences shown above the murine protein sequence represent peptides that were used for immunization.
Fig. 2. Immunoaffinity purification of canine Mtj1p from pancreatic microsomes. The post-ribosomal supernatant, derived from a detergent extract of microsomes, was incubated with antibody resin as described in Materials and methods. The specifically bound material was eluted with peptide. Aliquots of the post-ribosomal supernatant and the pass-through as well as 80 times larger aliquots of the last washing step and the eluate were subjected to electrophoresis in polyacrylamide gels and subsequent staining with Coomassie Blue (A) or subsequent electroblotting to PVDF membranes, followed by immunological detection (B). Minor amounts of BiP were detected in washing buffer and eluate, probably due to the known ability of this protein to bind to denatured proteins and peptides.
Fig. 3. Protease sensitivity of canine Mtj1p in dog pancreas microsomes. Aliquots of dog pancreas microsomes (RM in A–D) or of ribosome-stripped dog pancreas microsomes (PKRM in D) were left untreated or were supplemented with proteinase K (PK, 170 µg/ml) (A and B) or with trypsin (0.1–0.7 µg/ml) (C and D). (A and B) Digestion was carried out in the absence or presence of Triton X-100 [TX, 0.2% (v/v)] as indicated. (C and D) Digestion was carried out in the absence or presence of EDTA (10 mM) as indicated. After incubation for 60 min at 18 (A and B) or 0°C (C and D), the protease was inhibited and the samples were subjected to SDS–PAGE. The PVDF membranes were incubated with antibodies against the J-domain (A), BiP (B) or the C-terminal peptide of Mtj1p (C and D). The bound antibodies were made visible by incubation with ECL and exposure to X-ray film. The silver precipitation was quantified by densitometry (C and D). The intensity, obtained after protease treatment, is given as a percentage of the intensity derived from the untreated sample. The protein ladder was run on the same gel.
Fig. 4. In vitro synthesis of murine Mtj1p and nascent Mtj1p polypeptide chains. Mtj1p (A and D) and nascent Mtj1p polypeptide chains corresponding to 334 (B) or 190 (C) N-terminal amino acid residues were synthesized in rabbit reticulocyte lysates in the absence or presence of dog pancreas microsomes (RM) and in the presence of [35S]methionine (A and B) or [3H]leucine (C and D) as described in Materials and methods. The precursors of Mtj1p, Mtj1p-334mer and Mtj1p-190mer, respectively, are indicated with their predicted membrane orientation (C, C-terminal and cytosolic domain; J, lumenal J-domain; M, ER membrane; N, N-terminus; arrowhead, predicted cleavage site for signal peptidase). (A and B) The translation reactions were divided into two aliquots. One aliquot was subjected to centrifugation (10 min, 12 000 g, 4°C). The resulting supernatants (S) and pellets (P) as well as the aliquots, corresponding to the complete translation reactions, were subjected to SDS–PAGE and fluorography. (C and D) The translation reactions were divided into three aliquots. One aliquot was left untreated, the second aliquot was supplemented with proteinase K (PK; 170 µg/ml) and the third aliquot was supplemented with proteinase K plus Triton X-100 (TX; 0.2%). After incubation for 60 min at 0°C, the protease was inhibited and the samples were subjected to SDS–PAGE and fluorography. Note that, intentionally, the restriction digestion of the plasmid within the coding region was incomplete, giving rise to two translation products, the full-length protein (Mtj1p) and the nascent polypeptide chain (C). SDS–PAGE included a sample containing a mixture of 14C-labeled molecular mass standard proteins. p, precursor form; m, mature form. Note that 19 kDa, deduced on the basis of 14C-labeled molecular mass standard proteins, corresponds to 16 kDa as compared with the protein ladder (Figure 3).
Fig. 5. Apparent affinity of GST–MtjJ-hybrid for BiP as measured by surface plasmon resonance spectroscopy. Monoclonal goat anti-GST antibodies were immobilized on a sensor chip CM5. GST–MtjJ-hybrid was bound to the immobilized antibodies in the measuring cell and GST was bound to the immobilized antibodies in the reference cell. At time zero, defined concentrations of recombinant BiP (2 µM in B) were passed over the chip in the presence of ATP (A) and in the absence or presence of ATP or ATPγS (B), respectively. Each BiP application was followed by application of running buffer. The response units were recorded as the difference between the measuring and reference cell dependent on time. Note that the residuals, representing the difference between measured and fitted data, showed an amplitude of up to 12% of the maximum signal and a non-random distribution.
Fig. 6. Mtj1p interacts with ribosomes. (A) A detergent extract of microsomal proteins, described in Materials and methods, was diluted to a final potassium chloride concentration of 100 mM and subjected to sucrose gradient centrifugation [linear gradient between 10 and 60% (w/v) in low salt extraction buffer without glycerol] for 90 min at 54 000 r.p.m. and 2°C (Beckman SW 55 rotor). The molar ratio between ribosomes and Mtj1p was ∼4:1. (B) A detergent extract, derived from PKRM by solubilization plus centrifugation as described in Materials and methods, was adjusted to a final potassium chloride concentration of 100 mM and incubated with or without ribosomes, derived from dog pancreas, for 15 min at 30°C (± ribosomes). The ratio between ribosomes and Mtj1p was ∼20:1. Subsequently, the mixture was subjected to sucrose gradient centrifugation as described above. (C) A peptide eluate (as shown in Figure 2) was diluted to a final potassium chloride concentration of 100 mM and incubated with ribosomes, derived from dog pancreas, for 15 min at 30°C (+ ribosomes). The ratio between ribosomes and Mtj1p was >20:1. Subsequently, the mixture was subjected to sucrose gradient centrifugation [linear gradient between 10 and 60% (w/v) in low salt extraction buffer without glycerol] for 120 min at 49 000 r.p.m. and 2°C (Beckman SW 55 rotor). After fractionation of the gradients, aliquots of the fractions were precipitated according to Wessel and Flügge (1984) and subjected to SDS–PAGE and subsequent electroblotting to PVDF membranes, followed by immunological detection.
Fig. 7. Mtj1p interacts with ribosomes at low ionic strength. A detergent extract of microsomal proteins, described in Materials and methods, was kept at 400 mM KCl or diluted to final potassium chloride concentrations of 100 or 200 mM. The molar ratio between ribosomes and Mtj1p was ∼4:1. After centrifugation for 30 min at 68 000 r.p.m. and 2°C (Beckman TLA 100.3 rotor), supernatants (S) and pellets (P) were subjected to SDS–PAGE and subsequent electroblotting to PVDF membranes, followed by immunological detection.
Fig. 8. Recombinant Mtj1p derivatives. The results of the ribosome-binding assays, shown in Figures 6 and 9, are indicated. HLH, helix– loop–helix or myb domain.
Fig. 9. Certain recombinant Mtj1p derivatives interact with ribosomes. (A–D) The indicated recombinant proteins were adjusted to potassium chloride and bovine serum albumin concentrations of 100 mM and 150 µg/ml, respectively, and incubated with or without ribosomes, derived from dog pancreas, for 15 min at 30°C (± ribosomes). The molar ratios between ribosomes and Mtj1p were ∼2:1. Subsequently, the mixtures were subjected to sucrose gradient centrifugation [linear gradient between 10 and 60% (w/v) in low salt extraction buffer without glycerol, adjusted to 33 µg/ml albumin] for 90 min at 54 000 r.p.m. and 2°C (Beckman SW 55 rotor). Note that in the analysis of MtjC-ΔC, lysozyme was used instead of albumin (B). After fractionation of the gradients, aliquots of the fractions were precipitated according to Wessel and Flügge (1984) and subjected to SDS–PAGE and subsequent protein staining with Coomassie Blue (B) or to SDS–PAGE and subsequent electroblotting to PVDF membranes, followed by immunological detection (A, C and D). The position of ribosomes in the gradients, as deduced from the presence of stained ribosomal proteins, is indicated.
Fig. 10. In vitro protein synthesis is inhibited by certain Mtj1p derivatives. Bovine pre-prolactin (A and B) and firefly luciferase (C–F) were synthesized in rabbit reticulocyte lysate (A–D, G and H) or E.coli (Ec) lysate (E and F) in the presence of [35S]methionine. (G and H) Prior to translation, the rabbit reticulocyte lysate was fractionated into ribosomes and post-ribosomal supernatant by centrifugation. Subsequently, the post-ribosomal supernatant was combined with the ribosomal pellet that corresponded to the same, the 2- or the 4-fold volume of reticulocyte lysate. The translation reactions were supplemented with buffer [20 mM HEPES–KOH pH 7.5, 500 mM KCl, 2 mM MgCl2, 0.65% CHAPS, 2 mM dithiothreitol (DTT)], purified proteins (final concentration: 0.18 µM) in the same buffer (A, C, E and G), or peptides (final concentration: 50 µM) in the same buffer (B, D, F and H). After various incubation times, aliquots of the complete translation reactions were subjected to SDS–PAGE. The gels were analyzed by phosphoimager analysis. The amount of full-length radiolabeled protein that was produced at the end of the buffer control reaction was set to 100% (A–F). The final concentrations of mammalian lysate and bacterial lysate, respectively, in the translation reactions were 10 (A), 15 (E), 20 (B, C, G and H), 30 (F) and 40% (D) (v/v). Note that the concentration of ribosomes in the rabbit reticulocyte-based translation reaction [40% lysate, (v/v)] is ∼0.3 µM. Furthermore, note that post-translational addition of MtjC-ΔC did not result in a reduced level of translation product as compared with the untreated translation reaction (data not shown). wt-17mer (ELLGRKKRERKKKTGSK); nh-17mer (ELLGRKPRERPKKTGSK); rc-17mer (ELLGREKRERKEKTGSK).
Fig. 11. Mtj1p can recruit BiP to ribosomes in solution. Recombinant BiP (25 pmol) and Mg-ATP (1 mM) were incubated in the absence or presence of peptide eluate (as shown in Figure 2), diluted to a final potassium chloride concentration of 100 mM, and in the simultaneous absence or presence of ribosomes (50 pmol), derived from dog pancreas, for 15 min at 30°C. The molar ratio between ribosomes and Mtj1p was >2:1. Subsequently, the mixture was layered onto a cushion (0.5 M sucrose in 40 mM HEPES–KOH, pH 7.5, 150 mM potassium acetate, 5 mM magnesium acetate, 2 mM DTT) and subjected to centrifugation for 90 min at 100 000 r.p.m. and 2°C (Beckman TLA 100 rotor). The pellets were subjected to SDS–PAGE and subsequent electroblotting to PVDF membranes, followed by immunological detection.
Fig. 12. Observed Mtj1p interactions. Ribosomes with nascent polypeptide chains protect Mtj1p against protease. Protection is abolished by puromycin or EDTA, i.e. ribosome dissociation. Mtj1p binds to ribosomes in detergent solution. The cytosolic domain of Mtj1p binds to ribosomes. Mtj1p recruits BiP to ribosomes in detergent solution. The lumenal J-domain of Mtj1p binds to BiP.
References
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