Erf4p and Erf2p Form an Endoplasmic Reticulum-associated Complex Involved in the Plasma Membrane Localization of Yeast Ras Proteins

Zhao, Lihong; Lobo, Sandra; Dong, Xiangwen; Ault, Addison; Deschenes, Robert J.

doi:10.1074/jbc.m209760200

Cited by 74 publications

(61 citation statements)

References 56 publications

(68 reference statements)

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“…When expressed in HEK-293 cells, DHHC9 and GCP16 were resistant to extraction from membranes with salt or high pH but were extracted with the detergent dodecyl maltoside (data not shown). This behavior is characteristic of integral membrane proteins and similar to that of yeast Erf2⅐Erf4 (36,37).…”

Section: Identification Of a Candidate Mammalianmentioning

confidence: 68%

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DHHC9 and GCP16 Constitute a Human Protein Fatty Acyltransferase with Specificity for H- and N-Ras

Swarthout

Lobo

Farh

et al. 2005

Journal of Biological Chemistry

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322

347

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Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H-and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 codistribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2⅐Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9⅐GCP16 exhibits substrate specificity, palmitoylating H-and N-Ras but not myristoylated G ␣i1 or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9⅐GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.

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Section: Identification Of a Candidate Mammalianmentioning

confidence: 68%

“…There is a high degree of overlap in the expression of transcripts for DHHC9 and GCP16 (Fig. 2C) (36,37). To determine whether DHHC9 and GCP16 have similar properties, the nature of their membrane association was assessed.…”

Section: Identification Of a Candidate Mammalianmentioning

confidence: 99%

DHHC9 and GCP16 Constitute a Human Protein Fatty Acyltransferase with Specificity for H- and N-Ras

Swarthout

Lobo

Farh

et al. 2005

Journal of Biological Chemistry

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322

347

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“…The C-terminal hypervariable domain and the CaaX box are necessary and sufficient for plasma membrane localization of Ras proteins (19,75). In addition, plasma membrane localization of K-Ras in mammalian cells and Ras proteins in yeast does not require the classical secretory pathway or a functional Golgi apparatus (2).…”

Section: Resultsmentioning

confidence: 99%

“…KRas is also directed to the plasma membrane through a Golgi apparatus-independent pathway (2). Like K-Ras, yeast Ras proteins localize to the plasma membrane in the absence of a functional classical secretory pathway (19,75).…”

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confidence: 99%

Plasma Membrane Localization of Ras Requires Class C Vps Proteins and Functional Mitochondria in Saccharomyces cerevisiae

Wang

Deschenes

2006

Molecular and Cellular Biology

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Ras proteins are synthesized as cytosolic precursors, but then undergo posttranslational lipid addition, membrane association, and subcellular targeting to the plasma membrane. Although the enzymes responsible for farnesyl and palmitoyl lipid addition have been described, the mechanism by which these modifications contribute to the subcellular localization of Ras is not known. Following addition of the farnesyl group, Ras associates with the endoplasmic reticulum (ER), where palmitoylation occurs in Saccharomyces cerevisiae. The subsequent translocation of Ras from the ER to the plasma membrane does not require the classical secretory pathway or a functional Golgi apparatus. Vesicular and nonvesicular transport pathways for Ras proteins have been proposed, but the pathway is not known. Here we describe a genetic screen designed to identify mutants defective in Ras trafficking in S. cerevisiae. The screen implicates, for the first time, the class C VPS complex in Ras trafficking. Vps proteins are best characterized for their role in endosome and vacuole membrane fusion. However, the role of the class C Vps complex in Ras trafficking is distinct from its role in endosome and vacuole vesicle fusion, as a mitochondrial involvement was uncovered. Disruption of class C VPS genes results in mitochondrial defects and an accumulation of Ras proteins on mitochondrial membranes. Ras also fractionates with mitochondria in wild-type cells, where it is detected on the outer mitochondrial membrane by virtue of its sensitivity to protease treatment. These results point to a previously uncharacterized role of mitochondria in the subcellular trafficking of Ras proteins.

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“…However, in the rat exocrine pancreas the ␤ subunit was not found on Golgi membranes, whereas various ␣ subunits were detected there (31). Meanwhile, a palmitoyltransferase for Ras was found in ER membranes (32,33). The involvement of the Golgi in G protein trafficking and the location of a relevant palmitoyltransferase remain to be further investigated.…”

mentioning

confidence: 99%

Heterotrimer Formation, Together with Isoprenylation, Is Required for Plasma Membrane Targeting of Gऔγ

Takida

Wedegaertner

2003

Journal of Biological Chemistry

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Nascent ␤ and ␥ subunits of heterotrimeric G proteins need to be targeted to the cytoplasmic face of the plasma membrane (PM) in order to transmit signals. We show that ␤ 1 ␥ 2 is poorly targeted to the PM and predominantly localized to endoplasmic reticulum (ER) membranes when expressed in HEK293 cells, but co-expression of a G protein ␣ subunit allows strong PM localization of the ␤ 1 ␥ 2 . Furthermore, C-terminal isoprenylation of the ␥ subunit is necessary but not sufficient for PM localization of ␤ 1 ␥ 2 . Isoprenylation of ␥ 2 and localization of ␤ 1 ␥ 2 to the ER occurs independently of ␣ expression. Efficient PM localization of ␤ 1 ␥ 2 in the absence of co-expressed ␣ is observed when a site for palmitoylation, a putative second membrane targeting signal, is introduced into ␥ 2 . When a mutant of ␣ s is targeted to mitochondria, ␤ 1 ␥ 2 follows, consistent with an important role for ␣ in promoting subcellular localization of ␤␥. Heterotrimeric G proteins 1 are composed of ␣ and ␤␥ subunits. The ␤␥ complex only dissociates when denatured and hence is a functional monomer under physiological conditions. Upon receptor activation the ␤␥ dimer is freed from GTP-bound ␣ and relays signals to downstream molecules until it reassociates with GDP-bound ␣, re-forming the heterotrimer. To perpetuate this G protein cycle, the trimer must be tethered to the cytoplasmic face of the PM. This crucial subcellular localization is promoted by the covalent attachment of lipids to the subunits. Three lipid modifications have been found in G proteins, namely myristoylation and/or palmitoylation for the ␣ subunit and isoprenylation for the ␥ subunit. Myristoylation is the covalent attachment of a 14-carbon saturated myristate to an N-terminal glycine through an amide bond, whereas palmitoylation is a 16-carbon saturated palmitate linked to a cysteine via a thioester bond. Isoprenylation is a lipid modification in which an unsaturated, 15-carbon farnesyl isoprenoid or 20-carbon geranylgeranyl isoprenoid is linked to a cysteine, via a thioether bond.Mechanisms underlying the PM targeting of the ␣ subunit have been studied in some detail (1-3). The available data suggest a model in which myristoylation and/or binding to ␤␥ subunits constitutes an initial membrane targeting signal for the ␣ subunit. Subsequently, palmitoylation functions as a second signal that specifies localization to the PM. In contrast to the ␣ subunit, relatively less is known about how ␤␥ is targeted to the PM. Either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is linked to a cysteine residue in the so-called CAAX motif in the C terminus of the ␥ subunit (4, 5). The CAAX box (where C is a cysteine, A is commonly an aliphatic amino acid, and X can be one of several amino acids) is a consensus sequence for isoprenylation. The X residue is thought to specify which isoprenoid group will be linked to the cysteine. Among 12 human ␥ subunits thus far identified, ␥ 1 , ␥ 9 , and ␥ 11 have serine in the X position and are farnesylated, and the rest of ...

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Erf4p and Erf2p Form an Endoplasmic Reticulum-associated Complex Involved in the Plasma Membrane Localization of Yeast Ras Proteins

Cited by 74 publications

References 56 publications

DHHC9 and GCP16 Constitute a Human Protein Fatty Acyltransferase with Specificity for H- and N-Ras

DHHC9 and GCP16 Constitute a Human Protein Fatty Acyltransferase with Specificity for H- and N-Ras

Plasma Membrane Localization of Ras Requires Class C Vps Proteins and Functional Mitochondria in Saccharomyces cerevisiae

Heterotrimer Formation, Together with Isoprenylation, Is Required for Plasma Membrane Targeting of Gऔγ

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