C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

Farnsworth, Christopher C.; Kawata, Masahito; Yoshida, Yasuhisa; Takai, Yoshimi; Gelb, Michael H.; Glomset, John A.

doi:10.1073/pnas.88.14.6196

Cited by 166 publications

(75 citation statements)

References 31 publications

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“…We also tested the interaction between C7 and Rab7 lacking only the last three C-terminal amino acids (Rab7⌬CSC). Such a deletion prevents post-translational prenylation at the C terminus (32) and consequently decreases the background (or false positives) in the two-hybrid assay (12). Deletion of the prenylation sequence did not abolish the interaction confirming the specificity of the interaction.…”

Section: Resultsmentioning

confidence: 87%

The Proteasome α-Subunit XAPC7 Interacts Specifically with Rab7 and Late Endosomes

Dong

Chen

Welford

et al. 2004

Journal of Biological Chemistry

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Rab7 is a key regulatory protein governing early to late endocytic membrane transport. In this study the proteasome ␣-subunit XAPC7 (also known as PSMA7, RC6 -1, and HSPC in mammals) was identified to interact specifically with Rab7 and was recruited to multivesicular late endosomes through this interaction. The protein interaction domains were localized to the C terminus of XAPC7 and the N terminus of Rab7. XAPC7 was not found on early or recycling endosomes, but could be recruited to recycling endosomes by expression of a Rab7-(1-174)Rab11-(160 -202) chimera, establishing a central role for Rab7 in the membrane recruitment of XAPC7. Although XAPC7 could be shown to associate with membranes bearing ubiquitinated cargo, overexpression had no impact on steady-state ubiquitinated protein levels. Most notably, overexpression of XAPC7 was found to impair late endocytic transport of two different membrane proteins, including EGFR known to be highly dependent on ubiquitination and proteasome activity for proper endocytic sorting and lysosomal transport. Decreased late endocytic transport caused by XAPC7 overexpression was partially rescued by coexpression of wild-type Rab7, suggesting a negative regulatory role for XAPC7. Nevertheless, Rab7 itself was not subject to XAPC7-dependent proteasomal degradation. Together the data establish the first direct molecular link between the endocytic trafficking and cytosolic degradative machineries.

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Section: Resultsmentioning

confidence: 87%

The Proteasome α-Subunit XAPC7 Interacts Specifically with Rab7 and Late Endosomes

Dong

Chen

Welford

et al. 2004

Journal of Biological Chemistry

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“…The CXC Rab proteins, but not the CC Rab proteins, are then carboxyl-methylated (12, 19 -23). For example, Rab3a and Rab4, which terminate in Cys-Ala-Cys and Cys-Gly-Cys, respectively, are carboxyl methylated (19,20), whereas Rab1A and Rab2, which terminate in Cys-Cys, are not (12, 23). 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 (12).…”

Section: Discussionmentioning

confidence: 99%

Isoprenylcysteine Carboxyl Methyltransferase Deficiency in Mice

Bergö

Leung

Ambroziak

et al. 2001

Journal of Biological Chemistry

153

124

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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 Icmtdeficient 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.After isoprenylation, Ras and other proteins that terminate with a CAAX 1 sequence undergo two additional C-terminal modifications (1). First, the last three amino acids of the protein (i.e. the -AAX) are released by an endoprotease associated with the endoplasmic reticulum (1-3). Second, the carboxyl group of the newly exposed isoprenylcysteine is methylated (4, 5). These post-isoprenylation processing steps may help target CAAX proteins to membrane surfaces within cells (1).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 (1). Several groups have hypothesized that inhibiting the endoprotease or the methyltransferase might retard the growth of tumors caused by mutation-activated Ras proteins (1, 2, 6, 7). 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 (8). Membranes from Rce1-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 bef...

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“…The post-translational modifications of Sec4/Yptl/rab proteins are less well understood, but these proteins do not seem to undergo proteolytic cleavage at their C-terminus and are geranyl-geranylated instead of farnesylated . A protein such as smg p25Nrab3A which bears a C-terminal sequence similar to that of rab6p (Cys-Xaa-Cys) has been shown to contain geranylgeranyl groups attached on both cysteines [44]. The membrane-attachment process of ras and ras-like GTP-binding proteins is likely to be a reversible phenomenon.…”

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confidence: 99%

Comparison of the biochemical properties of unprocessed and processed forms of the small GTP‐binding protein, rab6p

Yang

Mollat

Chaffotte

et al. 1993

European Journal of Biochemistry

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The rab6 protein (rab6p) belongs to a large family of ras-like low-molecular-mass GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. When expressed in the baculovirus/insect cell system, two major forms of rab6p are obtained; a 24-kDa cytosolic unprocessed form and a 23-kDa membrane-bound form which represents the processed lipid-modified protein. Here, we have purified both forms to homogeneity and we have studied and compared their biochemical properties. Unprocessed and processed rab6p display similar bindingrate constants (k,,") for GDP and GTP (2-1.9 pM-' min-'). However, significant differences exist in the dissociation constants of bound guanine nucleotides. Processed rab6p in low and high magnesium solutions displays similar koff values for GTP and GDP. However, unprocessed rab6p has a ken, value higher for GDP than for GTP in both low and high magnesium solutions. Their intrinsic GTPase activities also differ ; unprocessed rab6p has an almost undetectable GTPase activity, whereas that of processed rab6p is in the same range as that reported for other ras and ras-like GTPbinding proteins (0.012 % 0.002 min-'). These results suggest that post-translational modifications of rab6p might induce subtle changes in the three-dimensional structure of the protein which affect the guanine-nucleotide-binding/hydrolysis activity.

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C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

Cited by 166 publications

References 31 publications

The Proteasome α-Subunit XAPC7 Interacts Specifically with Rab7 and Late Endosomes

The Proteasome α-Subunit XAPC7 Interacts Specifically with Rab7 and Late Endosomes

Isoprenylcysteine Carboxyl Methyltransferase Deficiency in Mice

Comparison of the biochemical properties of unprocessed and processed forms of the small GTP‐binding protein, rab6p

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