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Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry.

ABSTRACT: The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild-type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.

SUBMITTER: Trujillo C

PROVIDER: S-EPMC4113555 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry.

Trujillo Carolina C Taylor-Parker Julian J Harrison Robert R Murphy John R JR

Molecular microbiology 20100414 4

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 ...[more]

PMID: 20398220

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Project description:The ability of hydrophilic residues to shift the transverse position of transmembrane (TM) helices within bilayers was studied in model membrane vesicles. Transverse shifts were detected by fluorescence measurements of the membrane depth of a Trp residue at the center of a hydrophobic sequence. They were also estimated from the effective length of the TM-spanning sequence, derived from the stability of the TM configuration under conditions of negative hydrophobic mismatch. Hydrophilic residues (at the fifth position in a 21-residue hydrophobic sequence composed of alternating Leu and Ala residues and flanked on both ends by two Lys) induced transverse shifts that moved the hydrophilic residue closer to the membrane surface. At pH 7, the dependence of the extent of shift upon the identity of the hydrophilic residue increased in the order: L < G approximately = Y approximately = T < R approximately = H < S < P < K < E approximately = Q < N < D. By varying pH, shifts with ionizable residues fully charged or uncharged were measured, and the extent of shift increased in the order: L < G approximately = Y approximately = H(o) approximately = T < E(o) approximately = R < S < P < K+ < Q approximately = D(o) approximately = H+ < N approximately = E- < D-. The dependence of transverse shifts upon hydrophilic residue identity was consistent with the hypothesis that shift magnitude is largely controlled by the combination of side chain hydrophilicity, ionization state, and ability to position polar groups near the bilayer surface (snorkeling). Additional experiments showed that shift was also modulated by the position of the hydrophilic residue in the sequence and the hydrophobicity of the sequence moved out of the bilayer core upon shifting. Combined, these studies show that the insertion boundaries of TM helices are very sensitive to sequence, and can be altered even by weakly hydrophilic residues. Thus, many TM helices may have the capacity to exist in more than one transverse position. Knowledge of the magnitudes of transverse shifts induced by different hydrophilic residues should be useful for design of mutagenesis studies measuring the effect of transverse TM helix position upon function.

Dataset Information

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry.

Publications

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry.

Similar Datasets

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