Eukaryotic cell determination of ExoS ADP-ribosyltransferase substrate specificity

Jennifer E. Fraylick, Elizabeth A Rucks, Deanne M. Greene, Timothy S. Vincent, Joan C. Olson

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Exoenzyme S (ExoS) ADP-ribosylates multiple low-molecular-mass G- (LMMG-) proteins in vitro. Identification of the in vivo substrate specificity of ExoS has been hindered by its bacterial contact delivery into eukaryotic cells and difficulties in identifying ADP-ribosylated proteins within cells. Two-dimensional electrophoresis comparisons of substrate modifications by ExoS in vitro to that following bacterial translocation into HT-29 epithelial cells identified Ras, Ral, and Rab proteins and Rac1 as in vivo substrates of ExoS ADPRT activity. Cellular fractionation studies identified a relationship between membrane association and efficiency of substrate modification. Moreover, Rac and Cdc42 relocalized to the membrane in response to ExoS. Comparisons of substrate modification to time of exposure to ExoS identified a progression of substrate modification, with Ras, RalA, and Rab5 modified first, followed by Rab8 and 11, then Rab7 and Rac1. The data support that intrinsic properties of LMMG-proteins and their subcellular localization are determinants of bacterially translocated ExoS substrate selectivity.

Original languageEnglish (US)
Pages (from-to)91-100
Number of pages10
JournalBiochemical and Biophysical Research Communications
Volume291
Issue number1
DOIs
StatePublished - Jan 1 2002

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ADP Ribose Transferases
Eukaryotic Cells
Substrate Specificity
GTP-Binding Proteins
Adenosine Diphosphate
ral GTP-Binding Proteins
Bacterial Translocation
HT29 Cells
Membranes
Substrates
Electrophoresis
Epithelial Cells
Molecular mass
rac1 GTP-Binding Protein
Proteins
Fractionation
In Vitro Techniques
Association reactions
exoenzyme S

Keywords

  • ADP-ribosylation
  • Exoenzyme S
  • HT-29 epithelial cells
  • LMMG-proteins
  • Pseudomonas aeruginosa
  • Two-dimensional electrophoresis
  • Type III secretion

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Eukaryotic cell determination of ExoS ADP-ribosyltransferase substrate specificity. / Fraylick, Jennifer E.; Rucks, Elizabeth A; Greene, Deanne M.; Vincent, Timothy S.; Olson, Joan C.

In: Biochemical and Biophysical Research Communications, Vol. 291, No. 1, 01.01.2002, p. 91-100.

Research output: Contribution to journalArticle

Fraylick, Jennifer E. ; Rucks, Elizabeth A ; Greene, Deanne M. ; Vincent, Timothy S. ; Olson, Joan C. / Eukaryotic cell determination of ExoS ADP-ribosyltransferase substrate specificity. In: Biochemical and Biophysical Research Communications. 2002 ; Vol. 291, No. 1. pp. 91-100.
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