Document Type

Article

Publication Date

6-1-2010

Department

Biological Sciences

School

Biological, Environmental, and Earth Sciences

Abstract

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. Copyright (C) 2010 John Wiley & Sons, Ltd.

Comments

This is the peer reviewed version of the following article: [Chemical inhibition of CaaX protease activity disrupts yeast Ras localization. Yeast pn/a-n/a (2010)], which has been published in final form at https://doi.org/10.1002/yea.1756. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions: https://authorservices.wiley.com/author-resources/Journal-Authors/licensing/self-archiving.html#3.

Publication Title

Yeast

Volume

27

Issue

6

First Page

327

Last Page

343

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