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Review
. 2008:438:367-89.
doi: 10.1016/S0076-6879(07)38026-9.

Genetic analyses of the role of RCE1 in RAS membrane association and transformation

Martin O Bergo  1 , Annika M Wahlstrom, Loren G Fong, Stephen G Young
Affiliations
Review

Genetic analyses of the role of RCE1 in RAS membrane association and transformation

Martin O Bergo et al. Methods Enzymol. 2008.

Abstract

Proteins terminating with a CAAX motif, such as the nuclear lamins and the RAS family of proteins, undergo post-translational modification of a carboxyl-terminal cysteine with an isoprenyl lipid--a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by an endoprotease of the endoplasmic reticulum. RCE1 is responsible for the endoproteolytic processing of the RAS proteins and is likely responsible for endoproteolytic processing of the vast majority of CAAX proteins. Prenylation has been shown to be essential for the proper intracellular targeting and function of several CAAX proteins, but the physiologic importance of the endoprotease step has remained less certain. Here, we will review methods that have been used to define the physiologic importance of the endoproteolytic processing step of CAAX protein processing.

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Figures

Fig 1
Fig 1
An accumulation of “methylatable” substrates in Rce1 −/− cells. Methylation of whole-cell extracts from primary fibroblasts derived from Zmpste24+/+, Zmpste24 −/−, Icmt+/+, Icmt −/−, Rce1+/+, and Rce1 −/− embryos. The cell extracts were incubated with S-adenosyl-l-[methyl-14C]methionine, membranes containing high levels of Ste14p, yeast membranes containing high levels of mouse Rce1, and yeast membranes containing high levels of mouse Zmpste24. The relative level of methylatable substrates in wild-type and knockout cells was assessed with a base hydrolysis/methanol diffusion assay. For the Zmpste24+/+ and Zmpste24 −/− cells, we observed identical results when membranes expressing Rce1 were left out of the reaction mixture. Reproduced, with permission from The Journal of Biological Chemistry (Leung et al., 2001).
Fig 2
Fig 2
Intracellular localization of Ras proteins in immortalized fibroblasts. Rce1 flx/flx cells express normal levels of Rce1, whereas Rce1 α/α fibroblasts are completely deficient in Rce1 expression. Cre adenovirus fully converted the Rce1flx/flx fibroblasts into Rce1 α/α fibroblasts. Fibroblasts were fractionated into cytosolic (S100) and membrane (P100) fractions; Ras proteins were immunoprecipitated and analyzed on western blots of sodium dodecyl sulfate-polyacrylamide gels with antibody Ab-4. Note higher molecular weight of the Ras proteins in Rce1 α/α fibroblasts. Reproduced, with permission from Molecular and Cellular Biology (Bergo et al., 2002a).
Fig 3
Fig 3
Southern blot assessment of the ratio of Rce1flx and Rce1α alleles during the growth of mixed cultures of Rce1flx/flx and Rce1α/α fibroblasts. Mixed cultures of Rce1 flx/flx and Rce1α/α cells were obtained by infecting immortalized Rce1flx/flx fibroblasts with lacZ adenovirus and Cre adenovirus. Southern blots showing the ratio of Rce1α and Rce1flx alleles at different passages in four independent experiments. These experiments show that the Rce1flx/flx cells, which express Rce1, grow more rapidly than the Rce1α/α fibroblasts, which lack Rce1 expression. Reproduced, with permission from Molecular and Cellular Biology (Bergo et al., 2002a).
Fig 4
Fig 4
Comparison of the ability of K-Ras–transformed immortalized Rce1α/α and Rce1flx/flx fibroblasts to form colonies in soft agar. Rce1flx/flx fibroblasts were infected with a K-Ras retrovirus, then treated with either lacZ adenovirus or Cre adenovirus. In Rce1flx/flx cells treated with Cre adenovirus, the Rce1 was fully deleted, generating Rce1α/α fibroblasts. In each experiment, the excision of Rce1 resulted in fewer colonies in soft agar (P = 0.029, P = 0.005; P = 0.09; P = 0.008). Reproduced, with permission from Molecular and Cellular Biology (Bergo et al., 2002a).
Fig 5
Fig 5
Dilated cardiomyopathy in Rce1 lx/α αMyhc-Cre+/o mice. A, Increased size of hearts from Rce1flx/α αMyhc-Cre+/o mice, compared with Rce1+/+αMyhc-Cre+/o controls. B, H&E-stained sections of an Rce1flx/α αMyhc-Cre+/o heart and an Rce1+/+αMyhc-Cre+/o heart, both from 7-month-old mice. The hearts from Rce1flx/α αMyhc-Cre+/o mice were invariably dilated. Note the organized left atrial thrombus (arrow) in the heart from the Rce1flx/α αMyhc-Cre+/o mouse. Reproduced, with permission from The Journal of Biological Chemistry (Bergo et al., 2004).
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