Chemical inhibition of CaaX protease activity disrupts yeast Ras localization.
Proteins possessing a C-terminal CaaX motif, such as the Ras GTPases, undergo extensive post-translational modification that includes attachment of an isoprenoid lipid, proteolytic processing and carboxylmethylation. Inhibition of the enzymes involved in these processes is considered a cancer-therapeutic strategy. We previously identified nine in vitro inhibitors of the yeast CaaX protease Rce1p in a chemical library screen (Manandhar et al., 2007). Here, we demonstrate that these agents disrupt the normal plasma membrane distribution of yeast GFP-Ras reporters in a manner that pharmacologically phenocopies effects observed upon genetic loss of CaaX protease function. Consistent with Rce1p being the in vivo target of the inhibitors, we observe that compound-induced delocalization is suppressed by increasing the gene dosage of RCE1. Moreover, we observe that Rce1p biochemical activity associated with inhibitor-treated cells is inversely correlated with compound dose. Genetic loss of CaaX proteolysis results in mistargeting of GFP-Ras2p to subcellular foci that are positive for the endoplasmic reticulum marker Sec63p. Pharmacological inhibition of CaaX protease activity also delocalizes GFP-Ras2p to foci, but these foci are not as strongly positive for Sec63p. Lastly, we demonstrate that heterologously expressed human Rce1p can mediate proper targeting of yeast Ras and that its activity can also be perturbed by some of the above inhibitors. Together, these results indicate that disrupting the proteolytic modification of Ras GTPases impacts their in vivo trafficking.
Manandhar
Surya P
SP
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Hildebrandt
Emily R
ER
Jacobsen
William H
WH
Santangelo
George M
GM
Schmidt
Walter K
WK
D020816:Amino Acid Motifs; D002462:Cell Membrane / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D043484:Proprotein Convertases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism*
D006360:Heat-Shock Proteins; D026901:Membrane Transport Proteins; D011480:Protease Inhibitors; C074215:SEC63 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; D043484:Proprotein Convertases; C491326:RCE1 protein, human; C105193:RCE1 protein, S cerevisiae; D008666:Metalloendopeptidases; C489014:RAS1 protein, S cerevisiae; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins