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Isoprenylcysteine Carboxyl Methyltransferase Deficiency in Mice*

  • Martin O. Bergo
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    From the Gladstone Institute of Cardiovascular Disease, the

    Cardiovascular Research Institute,
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  • Gordon K. Leung
    Affiliations
    From the Gladstone Institute of Cardiovascular Disease, the

    Cardiovascular Research Institute,

    Department of Medicine, University of California, San Francisco, California 94141-9100; the
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  • Patricia Ambroziak
    Affiliations
    From the Gladstone Institute of Cardiovascular Disease, the
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  • James C. Otto
    Affiliations
    **Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710-3686;
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  • Patrick J. Casey
    Affiliations
    **Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710-3686;
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  • Anita Q. Gomes
    Footnotes
    Affiliations
    Molecular Genetics Section, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, London, SW7 2AZ, United Kingdom
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  • Miguel C. Seabra
    Affiliations
    Molecular Genetics Section, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, London, SW7 2AZ, United Kingdom
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  • Stephen G. Young
    Affiliations
    From the Gladstone Institute of Cardiovascular Disease, the

    Cardiovascular Research Institute,

    Department of Medicine, University of California, San Francisco, California 94141-9100; the
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants HL41633 and AG15451 (to S. G. Y.) and GM46372 (to P. J. C), a Wellcome Trust Programme Grant (to M. C. S.), and grant awards from the University of California Tobacco-related Disease Research Program (to M. O. B. and S. G. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    §§ Recipient of a Ph.D. Student Award from Fundação Ciência e Tecnologia of Portugal.
      After isoprenylation, Ras and other CAAX proteins undergo endoproteolytic processing by Rce1 and methylation of the isoprenylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). We reported previously thatRce1-deficient mice died during late gestation or soon after birth. We hypothesized that Icmt deficiency might cause a milder phenotype, in part because of reports suggesting the existence of more than one activity for methylating isoprenylated proteins. To address this hypothesis and also to address the issue of other methyltransferase activities, we generatedIcmt-deficient mice. Contrary to our expectation,Icmt deficiency caused a more severe phenotype thanRce1 deficiency, with virtually all of the knockout embryos (Icmt−/−) dying by mid-gestation. An analysis of chimeric mice produced from Icmt−/− embryonic stem cells showed that the Icmt−/− cells retained the capacity to contribute to some tissues (e.g. skeletal muscle) but not to others (e.g. brain). Lysates from Icmt−/− embryos lacked the ability to methylate either recombinant K-Ras or small molecule substrates (e.g. N-acetyl-S-geranylgeranyl-l-cysteine). In addition, Icmt−/− cells lacked the ability to methylate Rab proteins. Thus, Icmt appears to be the only enzyme participating in the carboxyl methylation of isoprenylated proteins.
      CAAX
      C-terminal motif consisting of Cys followed by two aliphatic residues (A) and any amino acid (X)
      Icmt
      isoprenylcysteine carboxyl methyltransferase
      ES
      embryonic stem
      AGGC
      N-acetyl-S-geranylgeranyl-l-cysteine
      AFC
      N-acetyl-S-farnesyl-l-cysteine
      GGPP
      geranylgeranylpyrophosphate
      REP
      Rab escort protein
      RabGGTase
      Rab geranylgeranyltransferase
      kb
      kilobases
      After isoprenylation, Ras and other proteins that terminate with a CAAX 1sequence undergo two additional C-terminal modifications (

      Young, S. G., Ambroziak, P., Kim, E., Clarke, S., The Enzymes, 3rd Ed., Tamanoi, F., Sigman, D. G., 21, 2001, 156, 213, Academic Press, San Diego, CA.

      ). First, the last three amino acids of the protein (i.e. the -AAX) are released by an endoprotease associated with the endoplasmic reticulum (

      Young, S. G., Ambroziak, P., Kim, E., Clarke, S., The Enzymes, 3rd Ed., Tamanoi, F., Sigman, D. G., 21, 2001, 156, 213, Academic Press, San Diego, CA.

      ,
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      ,
      • Schmidt W.K.
      • Tam A.
      • Fujimura-Kamada K.
      • Michaelis S.
      ). Second, the carboxyl group of the newly exposed isoprenylcysteine is methylated (
      • Clarke S.
      • Vogel J.P.
      • Deschenes R.J.
      • Stock J.
      ,
      • Dai Q.
      • Choy E.
      • Chiu V.
      • Romano J.
      • Slivka S.R.
      • Steitz S.A.
      • Michaelis S.
      • Philips M.R.
      ). These post-isoprenylation processing steps may help target CAAXproteins to membrane surfaces within cells (

      Young, S. G., Ambroziak, P., Kim, E., Clarke, S., The Enzymes, 3rd Ed., Tamanoi, F., Sigman, D. G., 21, 2001, 156, 213, Academic Press, San Diego, CA.

      ).
      The endoprotease and methyltransferase steps have attracted interest because they offer a potential means for modulating the activity of CAAX proteins, many of which participate in cell signaling (

      Young, S. G., Ambroziak, P., Kim, E., Clarke, S., The Enzymes, 3rd Ed., Tamanoi, F., Sigman, D. G., 21, 2001, 156, 213, Academic Press, San Diego, CA.

      ). Several groups have hypothesized that inhibiting the endoprotease or the methyltransferase might retard the growth of tumors caused by mutation-activated Ras proteins (

      Young, S. G., Ambroziak, P., Kim, E., Clarke, S., The Enzymes, 3rd Ed., Tamanoi, F., Sigman, D. G., 21, 2001, 156, 213, Academic Press, San Diego, CA.

      ,
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      ,
      • Clarke S.
      ,
      • Otto J.C.
      • Kim E.
      • Young S.G.
      • Casey P.J.
      ). At this point, however, testing such hypotheses appears to be a few years away. No specific high affinity inhibitors suitable for animal testing have been developed, either for the endoprotease or for the methyltransferase. Just as importantly, neither the spectrum of substrates nor the physiologic importance of the two processing steps has been explored fully. This is particularly the case for the methyltransferase.
      To define the physiologic relevance of the post-isoprenylation processing steps, our laboratory generated and characterized mice lacking the endoprotease Rce1 (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      ). Membranes fromRce1-deficient embryos and cells were completely unable to carry out the endoproteolytic processing of Ras and a host of other CAAX proteins. Surprisingly, the consequences of knocking out Rce1 in the mouse were relatively mild. Although most of the Rce1 knockout mice died before birth, the embryos remained viable until late in gestation, and as late as embryonic day 18.5 many were normal in size, appeared healthy, and had no obvious histologic abnormalities. A few of the Rce1 knockout mice were born and lived for a few weeks.
      A methyltransferase for mammalian CAAX proteins, isoprenylcysteine carboxyl methyltransferase (Icmt), has been identified recently (
      • Dai Q.
      • Choy E.
      • Chiu V.
      • Romano J.
      • Slivka S.R.
      • Steitz S.A.
      • Michaelis S.
      • Philips M.R.
      ) and shown to be located in the endoplasmic reticulum (
      • Dai Q.
      • Choy E.
      • Chiu V.
      • Romano J.
      • Slivka S.R.
      • Steitz S.A.
      • Michaelis S.
      • Philips M.R.
      ,
      • Choy E.
      • Chiu V.K.
      • Silletti J.
      • Feoktistov M.
      • Morimoto T.
      • Michaelson D.
      • Ivanov I.E.
      • Philips M.R.
      ). We used gene-targeting techniques to produce a mouse embryonic stem (ES) cell line lacking both Icmt alleles and documented that membranes from those cells lacked the ability to methylate recombinant K-Ras (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.C.
      • Young S.G.
      ). It is important to note, however, that existing reports have raised the possibility that certain isoprenylated proteins might be methylated by other enzymatic activities, at least in some cell types. For example, Giner and Rando (
      • Giner J.-L.
      • Rando R.R.
      ) concluded that there were distinct methyltransferase activities for the two classes of carboxyl-methylated isoprenylated proteins, the CAAX proteins and the CXC Rab proteins (
      • Giner J.-L.
      • Rando R.R.
      ). That conclusion was based on a variety of reciprocal inhibition studies with different methyltransferase substrates and inhibitors.
      The goal of the current study was to generate Icmt knockout mice, both to define the physiologic consequences of Icmtdeficiency and to further define biochemical roles of Icmt. Based in part on the report of an additional methyltransferase activity (
      • Giner J.-L.
      • Rando R.R.
      ), our a priori prediction was that Icmt-deficient mice might be affected less severely than the Rce1-deficient mice and might even be viable. We also predicted thatIcmt-deficient cells might retain the capacity to methylate the CXC-containing Rab proteins. As outlined in this report, both of those expectations were dashed by our experimental results.

      MATERIALS AND METHODS

       Generation of Icmt-deficient Mice

      A sequence replacement gene-targeting vector designed to replace exon 1 of the mouseIcmt gene with a neomycin-resistance gene (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.C.
      • Young S.G.
      ) was electroporated into 129/SvJae ES cells, and targeted cells (Icmt+/−) were identified on Southern blots with a 5′-flanking probe (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.C.
      • Young S.G.
      ). Two clones, each with a single neointegration, were used to produce Icmt+/− mice. Timed matings of Icmt+/− mice were performed to assess the viability of homozygotes (Icmt−/−) at different stages of development. The genotype of each embryo was determined by Southern blot analysis (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.C.
      • Young S.G.
      ). Icmt−/− fibroblasts were produced from mouse embryos as previously described (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      ).

       Production of Chimeric Mice from Icmt−/− ES Cells

      To assess the contribution of Icmt-deficient ES cells to different tissues, two independent lines of Icmt−/− ES cells (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.C.
      • Young S.G.
      ) were injected into C57BL/6 blastocysts. Ten male chimeric mice were obtained; all were 35–75% chimeras as judged by coat color. At 8 weeks of age, the mice were sacrificed, and genomic DNA was purified from multiple tissues and analyzed by Southern blot with a 32P-labeled Icmt probe. The ratio ofIcmt− to Icmt+ bands in each tissue was determined by phosphorimager.

       Measurement of Icmt Activity in Embryo Lysates

      Embryos were harvested and immediately placed in ice-cold buffer A (50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 1 mm EDTA, 100 mm NaCl), supplemented with a protease inhibitor mixture (Complete Mini, Roche Molecular Biochemicals). The embryos were homogenized with a Polytron and then centrifuged at 500 × g for 5 min to remove debris. The protein concentration of the homogenate was determined with a Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA). To measure Icmt activity, lysates (40–100 μg) were incubated with 10 μm S-adenosyl-l-[methyl-14C]methionine (55 Ci/mol, Amersham Pharmacia Biotech) and 50 μm of eitherN-acetyl-S-geranylgeranyl-l-cysteine (AGGC, Biomol) orN-acetyl-S-farnesyl-l-cysteine (AFC, Biomol). Recombinant farnesyl-K-Ras (
      • Otto J.C.
      • Kim E.
      • Young S.G.
      • Casey P.J.
      ) and recombinant geranylgeranyl-Rab proteins (described below) were also tested as methyl-accepting substrates. The total volume for the methylation reactions was 50 μl. After a 30-min incubation at 37 °C, the methylation reaction was stopped by adding 50 μl of 1.0 mNaOH containing 0.1% SDS. Most of the reaction mixture (90 μl) was spotted onto a pleated 2 × 8-cm filter paper wedged in the neck of a 20-ml scintillation vial containing 5 ml of scintillation fluid (ScintiSafe Econo 1, Fisher). The vials were capped and incubated at room temperature for 5 h to allow the [14C]methanol (formed by base hydrolysis of methyl esters) to diffuse into the scintillation fluid (
      • Clarke S.
      • Vogel J.P.
      • Deschenes R.J.
      • Stock J.
      ). The filter papers were then removed, and the vials were counted for radioactivity. Methyltransferase activity (pmol/mg total cell protein/min) was calculated after subtracting the background level of methylation in control reactions (lysates andS-adenosyl-l-[methyl-14C]methionine but no isoprenylated substrates). For the Rab methylation assays, control reactions also contained Rab1A, a CC Rab protein that does not undergo carboxyl methylation (
      • Smeland T.E.
      • Seabra M.C.
      • Goldstein J.L.
      • Brown M.S.
      ).

       Expression and Purification of Recombinant Rab Proteins

      Recombinant Rab proteins, Rab escort protein (REP), and Rab geranylgeranyltransferase (RabGGTase) were purified as previously described (
      • Armstrong S.A.
      • Brown M.S.
      • Goldstein J.L.
      • Seabra M.C.
      ,
      • Cremers F.P.M.
      • Armstrong S.A.
      • Seabra M.C.
      • Brown M.S.
      • Goldstein J.L.
      ,
      • Seabra M.C.
      • James G.L.
      ). Briefly, bovine Rab3B, murine Rab3D, and human Rab6 were expressed as N-terminal histidine-tagged fusion proteins inEscherichia coli with pET14b (Novagen, Madison, WI) as the expression vector for Rab3B and Rab6 and pRSET (Invitrogen, Carlsbad, CA) for Rab3D. The proteins were purified by affinity chromatography using a Ni2+-Sepharose resin. RabGGTase was expressed in Sf9 insect cells by coinfection with baculoviruses coding for both the α- and β-subunits and then purified by cation exchange chromatography followed by gel filtration chromatography. REP was also expressed in Sf9 cells as a C-terminal histidine-tagged fusion protein and was purified by Ni2+-Sepharose affinity chromatography.

       Preparation of Gernanylgeranylated REP·Rab Complexes

      Geranylgeranylated Rab·REP complexes (REP·RabGG) were formed in vitro by incubating Rab proteins with RabGGTase, and geranylgeranylpyrophosphate (GGPP) in the presence of limiting amounts of REP (

      Pereira-Leal, J. B., Gomes, A. Q., and Seabra, M. C. (2001) Methods Mol. Biol., in press.

      ). Rab protein (10 μm) was incubated with 2.5 μm REP1, 0.7 μmRabGGTase, and 20 μm unlabeled GGPP (Sigma) in a 50 mm sodium HEPES buffer, pH 7.2, containing 5 mmMgCl2, 1 mm dithiothreitol, and 0.05 mm protein-grade Nonidet P-40 (Calbiochem). The 50 μl reaction was incubated at 37 °C for 45 min. The complexes were stored at 4 °C with 1 mg/ml of bovine serum albumin as a carrier protein. To determine the efficiency of geranylgeranylation, parallel reactions were performed in the presence of [3H]GGPP ([1-3H] all-trans-GGPP, 15–30 Ci/mmol, PerkinElmer Life Sciences), and the incorporated [3H]GG was measured by scintillation after filtration through glass fiber filters (
      • Seabra M.C.
      • James G.L.
      ).

       Northern Blot Analysis

      A 262-base pair32P-labeled Icmt cDNA probe (spanning exons 1–3 of the Icmt gene) was hybridized to a mouse multiple-tissue poly(A)+ RNA blot (CLONTECH, Palo Alto, CA); hybridization and washing were performed as described previously (
      • Otto J.C.
      • Kim E.
      • Young S.G.
      • Casey P.J.
      ). The blot was exposed to x-ray film for 72 h at −80 °C.

       Quantification of Substrate Accumulation in Icmt−/− cells

      To assess the level of methylation substrates within cells, whole-cell lysates (Icmt+/+, Icmt+/−, andIcmt−/−) were incubated withS-adenosyl-l-[methyl-14C]methionine and recombinant Ste14p (10 μg of membrane protein from Sf9 cells that overexpress yeast STE14) (
      • Otto J.C.
      • Kim E.
      • Young S.G.
      • Casey P.J.
      ). The amount of base-labile methylation was quantified as described above.

      RESULTS

       Biochemical Analysis of Icmt-deficient Embryos

      Icmt+/− mice were produced from two independent lines of ES cells. Genotyping of 21-day-old offspring fromIcmt+/− intercrosses revealed that about two-thirds (58 of 82) were heterozygotes, and the remainder were wild-type. Genotyping of embryos revealed that Icmt−/− embryos constituted 25% of the litter until embryonic day 10.5 (E10.5, Fig.1 B). By E11.5, there were only a few viable Icmt−/− embryos; those embryos had beating hearts and red blood cells but were far smaller than heterozygous and wild-type mice (Fig. 1 C). Virtually all of theIcmt−/− mice died by E12.5, several days before the first of the homozygous Rce1 knockout embryos started to die (
      • Kim E.
      • Ambroziak P.
      • Otto J.C.
      • Taylor B.
      • Ashby M.
      • Shannon K.
      • Casey P.J.
      • Young S.G.
      ). Histologic studies of Icmt−/− embryos did not reveal a specific cause of death.
      Figure thumbnail gr1
      Figure 1Death of Icmt −/− embryos at mid-gestation. A, Southern blot of the genomic DNA from the yolk sacs of Icmt+/+, Icmt+/−, andIcmt−/− embryos. Genomic DNA was digested withBamHI. The Southern blot was hybridized with a 5′ probe (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.C.
      • Young S.G.
      ). The BamHI fragment in the wild-type allele is 5.0 kb, whereas it is 6.8 kb in the targeted allele.B, percentages of Icmt+/+ (●),Icmt+/− (♦), and Icmt−/− (▪) embryos surviving at different time points. C, Icmt+/+,Icmt+/−, and Icmt−/− embryos at E11.5.
      To gain insights into the importance of Icmt in the formation of different organs, we generated male chimeric mice (n = 10) with two lines of Icmt−/− ES cells (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.C.
      • Young S.G.
      ) and performed Southern blots to assess the relative capacities of Icmt+/+ and Icmt−/− cells to populate different tissues (Fig.2 A). The Icmt−/− cells contributed significantly to the development of skeletal muscle, as shown by the 1:1 ratio of Icmt− and Icmt+ band intensities in the genomic DNA of that tissue but made a negligible contribution to the formation of the brain. TheIcmt− band was virtually undetectable in brain, and theIcmt+/Icmt− band ratio was about 8:1. HighIcmt+/Icmt− ratios were also present in liver and testis. Interestingly, the extent to which Icmt−/− cells contributed to the formation of each tissue appeared to be inversely correlated with the normal levels of Icmtexpression in that tissue. Thus, as documented by a Northern blot (Fig.2 B), Icmt expression in wild-type mice was quite low in skeletal muscle, but high in the brain and liver. Measurements of enzyme activity in wild-type mice were in general agreement with the Northern blot results, with high activity levels in the brain, testis, and liver and low levels in skeletal muscle (Fig. 2 C).
      Figure thumbnail gr2
      Figure 2Analyzing Icmt expression in different tissues. A, ratio ofIcmt+/Icmt− alleles in the organs of chimeric mice (n = 10) generated with Icmt−/− ES cells. The DNA was prepared from multiple tissues, digested withBamHI, and analyzed on Southern blots with anIcmt probe (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.C.
      • Young S.G.
      ). The ratio of the 5.0-kb Icmt+ band to the 6.8-kb Icmt− band for each organ (mean ± S.D.) is shown. B, Icmt expression inIcmt+/+ tissues assessed with a CLONTECH mouse multiple tissue Northern blot. C, ability of lysates from tissues of Icmt+/+ mice to methylateN-acetyl-S-geranylgeranyl-l-cysteine. Methylation (pmol/mg cell protein/min) was measured with a base-hydrolysis assay (
      • Bergo M.O.
      • Leung G.K.
      • Ambroziak P.
      • Otto J.C.
      • Casey P.C.
      • Young S.G.
      ). Bars show mean ± S.D.
      To determine whether Icmt−/− embryos retained the capacity to methylate isoprenylated proteins, perhaps through a redundant enzymatic activity, we tested the capacity of lysates ofIcmt−/− embryos to methylate farnesyl-K-Ras (Fig.3 A). No enzymatic activity above background levels was identified. We considered the possibility that a redundant enzymatic activity might not be able to methylate K-Ras and therefore tested the ability of Icmt−/− lysates to methylate two small molecule substrates,N-acetyl-S-geranylgeranyl-l-cysteine andN-acetyl-S-farnesyl-l-cysteine (Fig. 3 B). The results of those studies were identical to the results of the K-Ras experiments, loss of activity in lysates ofIcmt−/− embryos. We also measured methyltransferase activity against small molecule substrates in lysates fromIcmt+/− embryos and tissues from adult Icmt+/− mice (liver, brain, and heart). Activities were invariably reduced by 50% (data not shown), not less than 50%, as would be the case if there were redundant methyltransferase activities.
      Figure thumbnail gr3
      Figure 3Methyltransferase activity in embryo lysates. A, ability of lysates from Icmt+/+,Icmt+/−, and Icmt−/− embryos to methylate farnesyl-K-Ras4B. B, methylation ofN-acetyl-S-geranylgeranyl-l-cysteine (AGGC) andN-acetyl-S-farnesyl-l-cysteine (AFC) by embryo lysates. Bars show the mean ± S.D. In some cases, the S.D. were low so that the error bar is not visible above the column. C, accumulation of methylation substrates in embryo lysates. Lysates (100 μg) fromIcmt+/+, Icmt+/−, and Icmt−/− embryos (n = 2) were mixed withS-adenosyl-l-[methyl-14C]methionine (10 μm) and yeast Ste14p. Methylation was assessed with a base hydrolysis assay. The bar graph shows the average of values for the two embryos of each genotype.
      Consistent with the apparent absence of a redundant methyltransferase activity, there was a substantial accumulation of methyltransferase substrates in lysates from Icmt−/− embryos (i.e. an accumulation of cellular proteins that could be methylated by the yeast ortholog of Icmt, Ste14p, Fig.3 C).
      An earlier study (
      • Giner J.-L.
      • Rando R.R.
      ) concluded that distinctS-adenosylmethionine-dependent methyltransferase activities were responsible for the methylation of the CAAXand CXC groups of isoprenylated proteins. That result would predict that lysates from Icmt−/− embryos would retain the ability to methylate CXC Rab proteins. This was not the case. Lysates from Icmt−/− cells were incapable of methylating three different CXC Rab proteins, although those proteins were readily methylated by lysates fromIcmt+/+ cells (Fig. 4). The importance of Rab methylation by Icmt remains obscure, but one obvious possibility is that it is important for membrane targeting. In preliminary experiments, we have used cell fractionation experiments and the expression of GFP·Rab fusions to assess the localization of Rab6 (a Golgi CXC Rab protein) in Icmt−/− andIcmt+/+ cells but did not observe noticeable differences (data not shown).
      Figure thumbnail gr4
      Figure 4Methylation of CXC Rab proteins by lysates from Icmt +/+ and Icmt −/− cells. Recombinant Rab3B, Rab3D, and Rab6 were geranylgeranylated and tested as methylation substrates with lysates from Icmt+/+ and Icmt−/− fibroblasts. Bars show the mean ± S.D.

      DISCUSSION

      Icmt catalyzes the formation of a carboxyl methyl ester on the isoprenylcysteine of CAAX proteins. The methylation reaction is the last of three sequential CAAX-box modifications and the most subtle, at least from the perspective of the primary structure of the protein. Methylation changes the molecular mass of the protein by a mere 14 daltons versus several hundred for both the isoprenylation and endoprotease steps. We had predicted thatIcmt-deficiency might produce a relatively mild phenotype. First, deletion of the methyltransferase gene in yeast (i.e. STE14) has little impact apart from its effect on the mating pheromone a-factor (
      • Hrycyna C.A.
      • Sapperstein S.K.
      • Clarke S.
      • Michaelis S.
      ), and a-factor apparently does not exist in mammals. Second, Rce1 deficiency produced a relatively mild phenotype, with some knockout mice surviving for a few weeks after birth. Thus, we expected that murine Icmt deficiency would produce a similarly mild phenotype, or perhaps even milder given the studies suggesting the existence of additional Icmt-like activities in mammalian cells (
      • Giner J.-L.
      • Rando R.R.
      ,
      • Philips M.R.
      • Staud R.
      • Pillinger M.
      • Feoktistov A.
      • Volker C.
      • Stock J.B.
      • Weissmann G.
      ). Our a prioriprediction was not upheld. Icmt deficiency yielded a more severe phenotype, with most Icmt−/− embryos dying between E10.5 and E11.5. Importantly, our biochemical studies with embryo lysates did not uncover a residual or redundant Icmt-like activity, and the lysates manifested a striking increase in Ste14p substrates.
      The Icmt−/− embryos probably died becauseIcmt-deficient cells failed to grow and contribute to the formation of various organs. Southern blots of tissues from chimeric mice generated with Icmt−/− cells revealed thatIcmt-deficient cells are severely defective in their capacity to contribute to the formation of certain organs (e.g. liver and brain) although they retained the ability to contribute to the formation of others (e.g. skeletal muscle).
      Although no Southern blots were performed on skin DNA, the percentage of brown fur on the chimeric animals suggested that Icmt-deficient ES cells are fairly robust in contributing to the formation of the hair follicles.
      We doubt that this finding was spurious, for several reasons. First, similar results were obtained with two lines of Icmt−/− ES cells. Second, there was a reasonably strong inverse correlation between normal levels ofIcmt expression and the ability of Icmt−/− ES cells to contribute to the formation of a tissue. Third, theIcmt chimeric mouse experiments were performed in parallel with studies with Zmpste24−/− ES cells,
      Zmpste24 is the mouse ortholog ofAFC1 (STE24) in Saccharomyces cerevisiae (
      • Boyartchuk V.L.
      • Ashby M.N.
      • Rine J.
      ,
      • Fujimura-Kamada K.
      • Nouvet F.J.
      • Michaelis S.
      ,
      • Tam A.
      • Nouvet F.J.
      • Fujimura-Kamada K.
      • Slunt H.
      • Sisodia S.S.
      • Michaelis S.
      ).
      which robustly populated all of the tissues of chimeric mice.
      G. Leung, M. Bergo, and S. Young, unpublished observations.
      Why were the developmental abnormalities more severe inIcmt−/− embryos than in Rce1−/− embryos? One possibility is simply that Icmt has more substrates than Rce1. Indeed, our experiments revealed for the first time that the CXC Rab proteins (which are not processed by Rce1) are methylated by Icmt. Rab proteins terminating in CXC and CC are geranylgeranylated at both cysteines (
      • Smeland T.E.
      • Seabra M.C.
      • Goldstein J.L.
      • Brown M.S.
      ). The CXC Rab proteins, but not the CC Rab proteins, are then carboxyl-methylated (
      • Smeland T.E.
      • Seabra M.C.
      • Goldstein J.L.
      • Brown M.S.
      ,
      • Li G.
      • Stahl P.D.
      ,
      • Farnsworth C.C.
      • Kawata M.
      • Yoshida Y.
      • Takai Y.
      • Gelb M.H.
      • Glomset J.A.
      ,
      • Newman C.M.H.
      • Giannakouros T.
      • Hancock J.F.
      • Fawell E.H.
      • Armstrong J.
      • Magee A.I.
      ,
      • Giannakouros T.
      • Newman C.M.H.
      • Craighead M.W.
      • Armstrong J.
      • Magee A.I.
      ,
      • Wei C.
      • Lutz R.
      • Sinensky M.
      • Macara I.G.
      ). For example, Rab3a and Rab4, which terminate in Cys-Ala-Cys and Cys-Gly-Cys, respectively, are carboxyl methylated (
      • Li G.
      • Stahl P.D.
      ,
      • Farnsworth C.C.
      • Kawata M.
      • Yoshida Y.
      • Takai Y.
      • Gelb M.H.
      • Glomset J.A.
      ), whereas Rab1A and Rab2, which terminate in Cys-Cys, are not (
      • Smeland T.E.
      • Seabra M.C.
      • Goldstein J.L.
      • Brown M.S.
      ,
      • Wei C.
      • Lutz R.
      • Sinensky M.
      • Macara I.G.
      ). Interestingly, replacement of the CXC terminus of Rab3a with CC abolishes methylation, whereas the opposite result is obtained when the CC terminus of Rab1A is replaced with a CXC sequence (
      • Smeland T.E.
      • Seabra M.C.
      • Goldstein J.L.
      • Brown M.S.
      ).
      Our current studies show that the methylation of the CXC Rab proteins is carried out by Icmt, the same methyltransferase that is responsible for CAAX protein methylation. Thus, Icmt almost certainly has more substrates than Rce1, which has no role in Rab protein processing. If these additional substrates (i.e. the Rab proteins) lie at the root of the more severe developmental defects in Icmt deficiency, one might predict that the methylation of Rab proteins has a significant impact on their function. In the case of Rab6, we did not observe a detectable effect of methylation on the intracellular localization of the protein, but we caution against overinterpreting those results. Those experiments did not assess other potential effects of Rab methylation such as effects on protein function or stability.
      A second potential explanation for the more severe developmental problems in the Icmt−/− embryos is that the positioning of the carboxylate anion on CAAX proteins has a profound influence on the binding of isoprenylated proteins to membranes, protein partners, or both. Both Rce1 and Icmtdeficiency eliminate the carboxyl methylation of CAAXproteins and thereby leave the protein with a C-terminal carboxylate anion rather than an α-methyl ester. However, the position of the carboxylate anion differs, being within the isoprenylcysteine residue in the setting of Icmt deficiency and located three amino acids downstream in the setting of Rce1 deficiency. Methylation itself is clearly important for membrane binding: methylatingN-acetyl-S-farnesyl-l-cysteine increases the partitioning of that molecule into the organic phase of an n-octanol/water mixture (
      • Parish C.A.
      • Rando R.R.
      ), and methylating farnesylated CAAX peptides increases their binding to synthetic liposomes (
      • Silvius J.R.
      • l'Heureux F.
      ). The impact of carboxylate anion positioning (i.e. on the prenylcysteine or on the end of the C-terminal tripeptide) on lipid-binding properties has not been studied in a similar fashion. However, it is easy to imagine that a more vicinal carboxylate anion (i.e. with Icmt deficiency) might be more potent in inhibiting the association of the isoprenyl lipid with membranes.
      The positioning of the carboxylate anion might also affect prenylation-dependent protein-protein interactions. Chenet al. (
      • Chen Z.
      • Otto J.C.
      • Bergo M.O.
      • Young S.G.
      • Casey P.J.
      ) have shown that the binding of K-Ras to microtubules is dependent on the structure of the C terminus. Farnesylated K-Ras binds to microtubules, but this binding is eliminated by the Rce1-mediated release of the last three amino acids. Binding to microtubules is restored by carboxyl methylation. Thus, in the case of K-Ras, the precise structure of the C terminus affects protein-protein interactions. These data, along with the aforementioned considerations regarding membrane binding, have made the “carboxylate anion positioning” hypothesis quite intriguing. Of course, the effect of carboxylate anion positioning could differ for different CAAX proteins.
      A third potential explanation for the more severe phenotype inIcmt-deficient mice compared with Rce1-deficient mice is that some CAAX proteins might undergo endoproteolytic processing in the absence of Rce1. According to this hypothesis, the milder phenotype of Rce1 deficiency could reflect the fact that Rce1 processes fewer CAAXprotein substrates than Icmt. This possibility is reasonable, given that a second yeast protein, Afc1p (Ste24p),3 assists Rce1p in the removal of the -AAX from the farnesylated mating pheromone a-factor. Thus far, however, there has been no direct demonstration that the murine ortholog of AFC1,Zmpste24,3 has any role in CAAXprotein processing (

      Young, S. G., Ambroziak, P., Kim, E., Clarke, S., The Enzymes, 3rd Ed., Tamanoi, F., Sigman, D. G., 21, 2001, 156, 213, Academic Press, San Diego, CA.

      ).
      The development and characterization of Icmt-deficient mice has clarified the role of Icmt in mouse development and in the processing of isoprenylated proteins. Just as importantly, these studies have suggested new hypotheses. For example, it will now be of interest to determine the importance of methylation in Rab protein function and to test the carboxylate anion positioning hypothesis. In addition, the production of Icmt-deficient fibroblasts opens the door to addressing the importance of carboxyl methylation in the transformation of fibroblasts by mutation-activated forms of the Ras proteins.

      Acknowledgments

      We thank Viktoria Gustafsson for technical assistance, Matthew Ashby and Steven Clarke for helpful discussions, and Stephen Ordway for criticisms of the manuscript.

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