Endoproteolytic processing of RhoA by Rce1 is required for the cleavage of RhoA by Yersinia enterocolitica outer protein T.
The bacterial toxin Yersinia outer protein T (YopT) is a cysteine protease that cleaves Rho GTPases immediately upstream of a carboxyl-terminal isoprenylcysteine. By clipping off the lipid anchor, YopT releases Rho GTPases from membranes, resulting in rounding up of mammalian cells in culture. The proteolytic activity of YopT depends on the isoprenylation of the cysteine within the carboxyl-terminal CaaX motif, a reaction carried out by geranylgeranyltransferase type I. The CaaX motif (where "a" indicates aliphatic amino acids) of Rho proteins undergoes two additional processing steps: endoproteolytic removal of the last three amino acids (i.e., -aaX) by Rce1 (Ras-converting enzyme 1) and methylation of the geranylgeranylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). In in vitro experiments, RhoA retaining -aaX cannot be cleaved by YopT. Nothing is known, however, about the influence of Rce1-mediated removal of -aaX on the activity of YopT in living cells. We hypothesized that Rce1-deficient mouse fibroblasts, in which the geranylgeranylated Rho proteins are not endoproteolytically processed, would be resistant to YopT. Indeed, this was the case. Microinjection of recombinant YopT into Rce1-deficient fibroblasts had no impact on the subcellular localization of RhoA and no impact on cell morphology. To determine if carboxyl methylation is also required for YopT action, we microinjected YopT into Icmt-deficient fibroblasts. In contrast to the results with Rce1-deficient cells, YopT cleaved RhoA and caused rounding up of the Icmt-deficient cells. Our data demonstrate that Rce1-mediated removal of -aaX from isoprenylated Rho GTPases is required for the proteolytic activity of YopT in living cells, whereas carboxyl methylation by Icmt is not.
Institute for Experimental and Clinical Pharmacology and Toxicology, Albert-Ludwigs-University of Freiburg, Albert-Str. 25, 79104 Freiburg, Germany.
Florian
F
Fueller
Martin O
MO
Bergo
Stephen G
SG
Young
Klaus
K
Aktories
Gudula
G
Schmidt
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D051379:Mice; D011485:Protein Binding; D017368:Protein Prenylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D015008:Yersinia enterocolitica; D015011:Yersinia pseudotuberculosis / Q000201:enzymology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; C116117:YopT protein, Yersinia; D010450:Endopeptidases; C491324:Rce1 protein, mouse; D003546:Cysteine Endopeptidases; D020741:rho GTP-Binding Proteins