Fatty Acylation of Two Internal Lysine Residues Required for the Toxic Activity of
            <i>Escherichia coli</i>
            Hemolysin

Stanley, Peter; Packman, Len C.; Koronakis, Vassilis; Hughes, Colin

doi:10.1126/science.7801126

Cited by 191 publications

(208 citation statements)

References 32 publications

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“…In cases where it has been studied, sites of palmitoylation in vivo and in vitro are the same (Bizzozero et al, 1987;Gutierrez and Magee, 1991;Stanley et al, 1994). In most cell-free systems, palmitoylating activity is dramatically reduced if membranes are pretreated by boiling (Berger and Schmidt, 1984;Slomiany et al, 1984), suggesting an enzymatic reaction.…”

Section: Introductionmentioning

confidence: 99%

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

Caron

1997

MBoC

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It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(a,3)-methylenediphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.

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Section: Introductionmentioning

confidence: 99%

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

Caron

1997

MBoC

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“…As an alternative, we chose to study porcine brain tubulin that was palmitoylated in a cell-free system (Caron, 1997). In support of this decision, studies of other proteins have shown that sites of palmitoylation in vivo and in vitro are equivalent (Bizzozero et al, 1987;Gutierrez and Magee, 1991;Stanley et al, 1994). Different methodologies for protein structure analysis have been used to identify palmitoylated amino acids (Papac et al, 1992;Weimbs and Stoffel, 1992;Bizzozero et al, 1994a;Hackett et al, 1994 (Ozols, 1984), necessitating the use of gel filtration on Sephadex LH60.…”

Section: Proteinsmentioning

confidence: 99%

“…Cysteine residues are the primary sites of palmitoylation. However, serine, threonine, and lysine residues may also be palmitoylated (for review, see Bizzozero et al, 1994b;Hackett et al, 1994;Stanley et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

Ozols

Caron

1997

MBoC

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As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3Hlpalmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of a-tubulin. 3H-labeled palmitoylated a-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to Veptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in a-tubulin. The palmitoylated residues in a-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.

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“…A previous study (Stanley et al, 1994) has shown that the two specific sites for HlyC-dependent acylation are located 288 and 414 residues, respectively, upstream of the C-terminal secretion signal. Involvement of the C terminus in the acylation reaction seems extremely unlikely, since HlyA lacking the N-terminal part containing the proposed amphipathic helices, or deletion of the Cterminal part, which contains the secretion signal, were still acylated normally in vitro (Stanley et al, 1994). Despite many detailed genetic and biochemical studies of the properties of HlyA and its interaction with membranes, no evidence has been presented in the literature to indicate that the C terminus of HlyA is directly involved in haemolytic activity.…”

Section: Discussionmentioning

confidence: 99%

Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin

et al. 2010

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Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of b-lactamase when the latter was fused to a Cterminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the b-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.

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Fatty Acylation of Two Internal Lysine Residues Required for the Toxic Activity of Escherichia coli Hemolysin

Cited by 191 publications

References 32 publications

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin

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