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Isoprenylcysteine carboxyl methyltransferase deficiency in mice
M O Bergo1, G K Leung, P Ambroziak
1Gladstone Institute of Cardiovascular Disease, the Cardiovascular Research Institute, University of California, San Francisco, California 94141-9100, USA. mbergo@gladstone.ucsf.edu
Abstract:
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 that Rce1-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 generated Icmt-deficient mice. Contrary to our expectation, Icmt deficiency caused a more severe phenotype than Rce1 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.
Insights
Isoprenylcysteine carboxyl methyltransferase (Icmt) deficiency causes embryonic lethality, indicating Icmt is essential for protein methylation and development. This contrasts with Rce1 deficiency, suggesting Icmt
Area of Science:
- Molecular and Cellular Biology
- Biochemistry
- Developmental Biology
Background:
- Ras and CAAX proteins require isoprenylation, endoproteolytic processing by Rce1, and methylation by Icmt.
- Previous studies showed Rce1-deficient mice exhibit late-gestation or perinatal lethality.
- The existence of alternative methyltransferase activities for isoprenylated proteins was hypothesized.
Purpose of the Study:
- To investigate the in vivo role of Icmt in protein methylation.
- To determine the phenotypic consequences of Icmt deficiency.
- To clarify the necessity of Icmt in the methylation of isoprenylated proteins.
Main Methods:
- Generation and analysis of Icmt-deficient (Icmt-/-) mice.
- Construction and analysis of chimeric mice using Icmt-/- embryonic stem cells.
- Biochemical assays to assess methylation of K-Ras, small molecule substrates, and Rab proteins in Icmt-/- embryo lysates.
Main Results:
- Icmt deficiency resulted in a more severe phenotype than Rce1 deficiency, with embryonic lethality by mid-gestation.
- Chimeric analysis revealed impaired contribution of Icmt-/- cells to certain tissues like the brain.
- Lysates from Icmt-/- embryos demonstrated a complete lack of carboxyl methylation activity for isoprenylated proteins.
Conclusions:
- Icmt is essential for the carboxyl methylation of isoprenylated proteins.
- Icmt deficiency leads to severe developmental defects and embryonic lethality.
- Icmt appears to be the sole enzyme responsible for this critical methylation step.
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