Myristoylation Cannot Functionally Replace the Isoprenylation of Rab5

Li, G.P.; Barbieri, M. Alejandro; Stahl, Philip D.

doi:10.1006/abbi.1995.1070

Cited by 7 publications

(5 citation statements)

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“…Thus only N ‐ myristoylation should be sufficient for the correct localization and functions of Arf GTPases. On the other hand, the substitution of isoprenylation by N ‐myristoylation also abolished the function of Rab5 (Li et al ., 1995). The reason for such a difference in the dependence on fatty acylation remains unknown.…”

Section: Discussionmentioning

confidence: 99%

Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana

et al. 2001

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Ara6 of Arabidopsis thaliana is a novel member of the Rab/Ypt GTPase family with unique structural features. It resembles Rab5 GTPases best, but lacks a large part of the C-terminal hypervariable region and the cysteine motif, and instead harbors an extra stretch of amino acid residues containing myristoylation and palmitoylation sites at the N-terminus. Ara6 is tightly associated with membranes and is expressed constitutively. In contrast, the conventional Rab5 ortholog, Ara7, is highly expressed only in actively dividing cells. Examination of green fluorescent protein (GFP)-tagged proteins indicates that both Ara6 and Ara7 are distributed on a subpopulation of endosomes and suggests their roles in endosomal fusion. The endosomal localization of Ara6 requires N-terminal fatty acylation, nucleotide binding and the C-terminal amino acid sequence coordinately. Proteins similar to Ara6 are found only in higher plants and thus represent a novel class of Rab GTPases regulating endocytic function in a plant- specific manner.

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Section: Discussionmentioning

confidence: 99%

Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana

et al. 2001

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“…Moreover, lysates from infected cells were still able to ADP-ribosylate recombinant His 6 -Rho added in vitro. When Sindbis infection was used in studies of basic cell functions, such as posttranslational processing (40,41) or programmed cell death (36), no cytotoxicity was reported. We observed that cell viability was, in fact, higher than with scrape loading (Ͼ90% versus 60 -70%).…”

Section: Discussionmentioning

confidence: 99%

Effect of Rho and ADP-ribosylation Factor GTPases on Phospholipase D Activity in Intact Human Adenocarcinoma A549 Cells

Meacci

Vasta

Moorman

et al. 1999

Journal of Biological Chemistry

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Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADPribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.Phospholipase D (PLD), 1 an important effector in receptormediated signal transduction pathways, catalyzes the hydrolysis of the most abundant membrane phospholipid, phosphatidylcholine, to generate choline and phosphatidic acid. In intact cells, choline is rapidly phosphorylated to phosphocholine, which plays a role in cell proliferation (1). Phosphatidic acid also serves as a second messenger in the regulation of secretion, DNA synthesis, and cell proliferation (2). PLD activity appears to be regulated, and can be activated by both phosphatidylinositol 4,5-bisphosphate (3) and phosphatidylinositol 1,4,5-trisphosphate (4) in vitro. Phosphatidylinositol 4,5-bisphosphate was essential for PLD activation in intact cells (5). Consistent with a regulatory role for protein kinase C (PKC), phorbol esters markedly activated PLD (6, 7), and PKC inhibitors abolished agonist-induced PLD activity (8) in some cells.Both native and recombinant PLD were activated b...

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“…In other studies, dsSIN recombinants have been used to express the Japanese encephalitis virus (21) and rubella virus (22) stuctural proteins, to deliver a single chain antibody for intracellular immunization against tick-borne encephalitis (23), to map the domain of GLUT-4, the insulin-regulatable glucose transporter, which is responsible for efficient intracellular sequestration (24), to study structure-function aspects of ras-like GTP-binding proteins involved in vesicular transport (25)(26)(27)(28)(29), and to probe the interplay between viral and cellular genes involved in apoptotic cell death (30,31).…”

Section: Replication and Packaging-competent Vectorsmentioning

confidence: 99%

Alphavirus-based expression vectors: strategies and applications.

Frolov

Hoffman

Prágai

et al. 1996

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

199

142

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Alphaviruses are positive-strand RNA viruses that can mediate efficent cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication machinery has been engineered for high-level expression of heterologous RNAs 1 and 2). Over the past 10 years, the alphavirus RNA replication and packaging machinery has been adapted for expression of heterologous RNAs and proteins in animal cells (for reviews, see refs. 3-6). As transient expression systems, alphaviruses offer several advantages. These include (i) a broad range of susceptible host cells including those of insect, avian, and mammalian origin; (ii) high levels of cytoplasmic RNA and protein expression without splicing; and (iii) the facile construction and manipulation of recombinant RNA molecules using full-length cDNA clones from which infectious RNA transcripts can be generated by in vitro transcription. Two principal strategies are being employed for expression of heterologous sequences: (i) engineering infectious recombinant RNAs that express additional subgenomic RNAs and (ii) replacement of the structural genes to produce self-replicating RNA "replicons" that can be packaged into infectious particles using defective helper RNAs or packaging cell lines. In addition, incorporation of heterologous ligands or receptors into the virion envelope may eventually allow targeting of engineered alphavirus RNAs to specific cell types. This overview briefly discusses the background, methodology, and applications of these alphavirus vector systems, which range from high-level protein production in cell culture to the induction of protective immunity in animals. (Fig; 1). This subgenomic RNA, which can accumulate to levels approaching 106 molecules per cell, is the mRNA for translation of the structural proteins. The synthesis of minus, plus, and subgenomic RNAs is temporally regulated via proteolytic processing of nonstructural polyprotein replicase components by a virus-encoded protease residing in the C-terminal region of nsP2 (8, 9).The structural proteins are initially translated as a polyprotein (NH2-C-E3-E2-6K-E1-COOH) that is processed coand posttranslationally to produce the mature products. Cleavage at the C-E3 site is mediated by a chymotrypsin-like protease activity residing in the C-terminal portion of the C protein. E3 and E2 are initially made as a precursor (called PE2 or P62) that is processed by a furin-like activity late during release of the virus from infected cells. Envelope glycoproteins El and PE2, separated by signal peptidase cleavages, form a heterodimer that migrates through the secretory pathway to the plasma membrane. In the cytoplasm, C-protein subunits complex with the genome RNA to form a nucleocapsid that matures by budding through the plasma membrane, acquiring a lipid bilayer envelope with embedded viral glycoproteins. Infectious Alphavirus cDNA ClonesStudies on the use of alphaviruses as vectors have required the recovery of infectious replication-competent ...

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Myristoylation Cannot Functionally Replace the Isoprenylation of Rab5

Cited by 7 publications

References 0 publications

Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana

Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana

Effect of Rho and ADP-ribosylation Factor GTPases on Phospholipase D Activity in Intact Human Adenocarcinoma A549 Cells

Alphavirus-based expression vectors: strategies and applications.

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