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The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex.

Ratts R, Zeng H, Berg EA, Blue C, McComb ME, Costello CE, vanderSpek JC, Murphy JR - J. Cell Biol. (2003)

Bottom Line: The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast.In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain.Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA. ratts@bu.edu

ABSTRACT
In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

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Identification of putative CTF(s) using MS. (A) Representative total ion chromatogram from an online capillary liquid chromatography mass spectrometric analysis of the in-gel tryptic digest after immunoprecipitation of the 84-kD band (Fig. 2 B and Fig. 5 B) from human partially purified CTFs using rabbit polyclonal anti-Hsp 90 antibodies. (B) Mass spectrum from LC-MS elution at time 19.5 to 20.5 min, as indicated by the shaded region in (A). Peaks are labeled with the m/z value, the charge state, the corresponding amino acid segment, and specification of the Hsp 90 isoform. (C) Tandem mass spectrum for m/z 575.984+ (redundant sequence from Hsp 90 α and β; see inset on B). Complementary a and b (NH2-terminal derived) as well as y (COOH-terminal derived) ions are labeled in the spectrum with m/z value and charge state. All observed a, b, and y ions are indicated in the peptide diagram. Data were analyzed using BioAnalyst™ (Applied Biosystems) reconstruction algorithms. For initial screening and searches, acquired mass values were compared with theoretical protein digests using the Mascot search engine (Matrix Science Ltd.).
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fig4: Identification of putative CTF(s) using MS. (A) Representative total ion chromatogram from an online capillary liquid chromatography mass spectrometric analysis of the in-gel tryptic digest after immunoprecipitation of the 84-kD band (Fig. 2 B and Fig. 5 B) from human partially purified CTFs using rabbit polyclonal anti-Hsp 90 antibodies. (B) Mass spectrum from LC-MS elution at time 19.5 to 20.5 min, as indicated by the shaded region in (A). Peaks are labeled with the m/z value, the charge state, the corresponding amino acid segment, and specification of the Hsp 90 isoform. (C) Tandem mass spectrum for m/z 575.984+ (redundant sequence from Hsp 90 α and β; see inset on B). Complementary a and b (NH2-terminal derived) as well as y (COOH-terminal derived) ions are labeled in the spectrum with m/z value and charge state. All observed a, b, and y ions are indicated in the peptide diagram. Data were analyzed using BioAnalyst™ (Applied Biosystems) reconstruction algorithms. For initial screening and searches, acquired mass values were compared with theoretical protein digests using the Mascot search engine (Matrix Science Ltd.).

Mentions: Tryptic peptides from “in-gel” digestion of individual protein bands resolved by SDS-PAGE were subjected to analysis by mass spectroscopy using matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) and nano-electrospray ionization (ESI) quadrupole/orthogonal TOF spectrometers (Jensen et al., 1999). Peptide maps and tandem MS sequence data allowed for the unequivocal identification of Hsp 90 (α and β) and TrR-1 in the partially purified CTF complex mixture from human T cells (Fig. 4; Table I). Importantly, the corresponding yeast homologues, Hsp 82 and TrR-1, as well as thioredoxin peroxidase, were identified in the partially purified CTF complex from yeast cells (Table I). The cumulative peptide coverage for each protein identified through LC-MS/MS sequencing was between 65 and 85% of the total protein (Table S1, available at www.jcb.org/cgi/content/full/jcb.200210028/DC1). Ions unassigned in the LC-MS/MS spectra were indicative of truncation, sequence variation, and/or post-translational modification.


The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex.

Ratts R, Zeng H, Berg EA, Blue C, McComb ME, Costello CE, vanderSpek JC, Murphy JR - J. Cell Biol. (2003)

Identification of putative CTF(s) using MS. (A) Representative total ion chromatogram from an online capillary liquid chromatography mass spectrometric analysis of the in-gel tryptic digest after immunoprecipitation of the 84-kD band (Fig. 2 B and Fig. 5 B) from human partially purified CTFs using rabbit polyclonal anti-Hsp 90 antibodies. (B) Mass spectrum from LC-MS elution at time 19.5 to 20.5 min, as indicated by the shaded region in (A). Peaks are labeled with the m/z value, the charge state, the corresponding amino acid segment, and specification of the Hsp 90 isoform. (C) Tandem mass spectrum for m/z 575.984+ (redundant sequence from Hsp 90 α and β; see inset on B). Complementary a and b (NH2-terminal derived) as well as y (COOH-terminal derived) ions are labeled in the spectrum with m/z value and charge state. All observed a, b, and y ions are indicated in the peptide diagram. Data were analyzed using BioAnalyst™ (Applied Biosystems) reconstruction algorithms. For initial screening and searches, acquired mass values were compared with theoretical protein digests using the Mascot search engine (Matrix Science Ltd.).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172777&req=5

fig4: Identification of putative CTF(s) using MS. (A) Representative total ion chromatogram from an online capillary liquid chromatography mass spectrometric analysis of the in-gel tryptic digest after immunoprecipitation of the 84-kD band (Fig. 2 B and Fig. 5 B) from human partially purified CTFs using rabbit polyclonal anti-Hsp 90 antibodies. (B) Mass spectrum from LC-MS elution at time 19.5 to 20.5 min, as indicated by the shaded region in (A). Peaks are labeled with the m/z value, the charge state, the corresponding amino acid segment, and specification of the Hsp 90 isoform. (C) Tandem mass spectrum for m/z 575.984+ (redundant sequence from Hsp 90 α and β; see inset on B). Complementary a and b (NH2-terminal derived) as well as y (COOH-terminal derived) ions are labeled in the spectrum with m/z value and charge state. All observed a, b, and y ions are indicated in the peptide diagram. Data were analyzed using BioAnalyst™ (Applied Biosystems) reconstruction algorithms. For initial screening and searches, acquired mass values were compared with theoretical protein digests using the Mascot search engine (Matrix Science Ltd.).
Mentions: Tryptic peptides from “in-gel” digestion of individual protein bands resolved by SDS-PAGE were subjected to analysis by mass spectroscopy using matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) and nano-electrospray ionization (ESI) quadrupole/orthogonal TOF spectrometers (Jensen et al., 1999). Peptide maps and tandem MS sequence data allowed for the unequivocal identification of Hsp 90 (α and β) and TrR-1 in the partially purified CTF complex mixture from human T cells (Fig. 4; Table I). Importantly, the corresponding yeast homologues, Hsp 82 and TrR-1, as well as thioredoxin peroxidase, were identified in the partially purified CTF complex from yeast cells (Table I). The cumulative peptide coverage for each protein identified through LC-MS/MS sequencing was between 65 and 85% of the total protein (Table S1, available at www.jcb.org/cgi/content/full/jcb.200210028/DC1). Ions unassigned in the LC-MS/MS spectra were indicative of truncation, sequence variation, and/or post-translational modification.

Bottom Line: The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast.In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain.Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA. ratts@bu.edu

ABSTRACT
In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

Show MeSH
Related in: MedlinePlus