Sticky fingers and CAAX boxes

Magee, Tony; Hanley, Michael R.

doi:10.1038/335114a0

Cited by 80 publications

(34 citation statements)

References 11 publications

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“…lb). The stability of membrane attachment may reflect the possible fatty acylation of a cysteine at the rab4 COOH terminus, which ends with a Cys-Gly-Cys prenylation signal (39)(40)(41) Because the anodally shifted pool contained both endosomes and lysosomes, we next separated these two organelles by centrifuging the shifted pool in a Percoli density gradient before SDS/PAGE (33). As shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

The small GTP-binding protein rab4 is associated with early endosomes.

Sluijs

Hull

Zahraoui

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

263

118

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Small GTP-binding proteins ofthe rab family have been implicated as playing important roles in controlling membrane traffic on the biosynthetic and endocytic pathways. We demonstrate that a distinct rab protein, rab4p, is associated with the population of early endosomes involved in transferrinreceptor recycling. An antibody to human rab4p was found to detect a doublet of -24-kDa proteins on immunoblots from various cell types. Seventy-five percent of these proteins were tightly membrane bound and could be released only by detergent treatment. Upon isolation of early endosomes, late endosomes, and lysosomes, by free-flow electrophoresis and Percoll density-gradient centrifugation, most (70%) of the rab4p was found to cofractionate with early endosomes and endocytic vesicles containing '2I-labeled transferrin. The rab proteins previously localized to the endoplasmic reticulum and/or Golgi apparatus were not found in these fractions. We also localized rab4p to transferrin-receptor-containing early endosomes by immunofluorescence after expression of rab4 cDNA. The association of rab4p with early endosomes and other vesicles involved in the intracellular transport of transferrin receptor suggests that rab4p may play a role in regulating the pathway of receptor recycling.

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Section: Methodsmentioning

confidence: 99%

The small GTP-binding protein rab4 is associated with early endosomes.

Sluijs

Hull

Zahraoui

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

263

118

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“…This two subunits encoded by these genes. This idea was further CysAAX sequence is termed the 'CAAX box' [5] and the FTase strengthened by the detection of a significant (approx. 30 %) catalyses the addition of a farnesyl group to the cysteine.…”

Section: Introductionmentioning

confidence: 99%

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

Gomez

Goodman

Tripathy

et al. 1993

Biochemical Journal

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Protein farnesyltransferase (FTase) catalyses the addition of a farnesyl group to a cysteine within the so-called ‘CAAX box’ at the C-terminus of various proteins. In the present paper we report purification of Saccharomyces cerevisiae FTase to near-homogeneity. This was accomplished by constructing a yeast strain overproducing FTase approx. 100-fold. The purified enzyme was a heterodimer of approx. 90 kDa and consisted of 43 kDa and 34 kDa subunits. The 43 kDa subunit was shown to be the product of the DPR1 gene by using antibody raised against baculovirus-produced DPR1 polypeptide. The purified enzyme required Mg2+, showed a pH optimum of 7.8 and was most active at 50 degrees C. The Km values for farnesyl pyrophosphate and GST-CIIS (glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein), KmFpp and KmGST CIIS, were 8.1 and 5.1 microM respectively. The enzyme was capable of farnesylating GST-CIIL (the same as GST-CIIS, except that the C-terminal serine is changed to leucine), a substrate protein for the enzyme geranylgeranyltransferase, although with a higher apparent Km than for GST-CIIS. Like its mammalian counterpart, yeast FTase activity was inhibited by peptides containing the C-terminal CAAX sequence (that is, one where C = cysteine, A = aliphatic amino acid and X = any amino acid). These results provide direct evidence for the idea that the yeast and mammalian FTases are structurally and functionally very similar.

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“…Because the greatest amino acid sequence divergence between Ras and RaplA is in the COOH-terminal domain (25% identity in the last 74 residues), we examined whether this RaplA-specific region abrogated transformation. For Ras proteins, the COOH terminus is essential for localizing the protein to the plasma membrane (42, 43), in large part because of an unusual series of modifications to the last four amino acids, which have been termed a CAAX motif (C, cysteine; A, aliphatic; X, other) (8,31,36). These modifications include attachment of a C15 (farnesyl) isoprenoid to the cysteine of the CAAX motif (4, 21), removal of the final three amino acids (18,20), and methylation of the newly exposed oa-carboxyl group (8,12,20).…”

mentioning

confidence: 99%

The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.

Buss¹,

Quilliam²,

Kato³

et al. 1991

Mol. Cell. Biol.

107

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Although the RaplA protein resembles the oncogenic Ras proteins both structurally and biochemically, RaplA exhibits no oncogenic properties. Rather, overexpression of RaplA can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and RaplA occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of RaplA were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of RaplA) in NIH 3T3 cells and a full-length human RaplA protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length RaplA, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid.In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the RaplA COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:RaplA protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of RaplA can support the interactions between H-Ras and membranes that are required for transformation.The RaplA protein (also known as Krev-1 or smg p21) was isolated on the basis of strong structural (50% amino acid identity) and biochemical similarities to Ras (24,35) and, independently, by its ability to reverse transformation caused by Ras proteins (27). Despite the many similarities between Ras and RaplA proteins, the biochemical basis for this reversion remains unknown. It is possible that the regions of sequence identity between RaplA and Ras allow RaplA, when expressed at high levels (27), to act as a competitor for regulatory or target proteins of Ras. This model has recently received very strong support from studies that show that RaplA can interfere in vitro with the activity of Ras-GTPase-activating protein (GAP) (17, 22), a protein which binds to and stimulates the GTPase activity of Ras, but which fails to affect the ability of RaplA to hydrolyze GTP (17,37). An alternate model suggests that RaplA causes suppression through a mechanism independent of Ras; this would suggest that regions specific to RaplA participate in interactions with proteins of an inhibitory pathway. This possibility is supported by evidence for a GAP-like protein that is unique to RaplA (25). Because the greatest amino acid sequence divergence between Ras and RaplA is in the COOH-terminal domain (25% identity in t...

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Sticky fingers and CAAX boxes

Cited by 80 publications

References 11 publications

The small GTP-binding protein rab4 is associated with early endosomes.

The small GTP-binding protein rab4 is associated with early endosomes.

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.

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