Detection of low molecular mass GTP‐binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells

Fournier, S.; Parulekar, M. R.; Trifaró, J. M.

doi:10.1016/0014-5793(89)81254-2

Cited by 30 publications

(11 citation statements)

References 29 publications

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“…Various GTP-binding proteins have been localized to secretory granules (12,(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45). Thus far, only Rab3 has been clearly shown to be involved in regulated secretion (34)(35)(36)43).…”

Section: Discussionmentioning

confidence: 99%

Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.

Chen

Holcomb

Moore

1993

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

139

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Small GTP-binding proteins of the YPT/ SEC4/Rab family have been shown to play an essential role in intracellular membrane trafficking. In mammals, Rab8 and RablO are the two small GTP-binding proteins identified so far that are closest to SEC4, an essential gene product involved in post-Golgi constitutive secretion in the yeast Saccharomyces cerevisiae. To study the loIzation of Rab proteins, we have expressed the cDNAs with an influenza virus hemagglutinin (HA) epitope tag at the N terminus. The feasibility ofthis method was tested by using yeast SEC4. HA-tagged SEC4 functionally complemented a temperature-sensitive sec4 mutant similarly to wild-ype SEC4, indicating that the modified protein retained functional integrity. Monoclonal antibody 12CA5, raised against the HA tag, was used to determine the expression and localization of HA-tagged proteins after transfection. In stably transfected CHO and Swiss 3T3 cells, HA-tagged Rab8 was localized to the cell periphery, with the highest concentration in the rufling areas. In contrast, epitope-tagged RablO expressed in CHO and BHK cells was concentrated on membranes in the perinuclear region. By light microscopy, the staining partially overlapped with that of a Golgi marker, 3-COP. Thus, despite the high degree homology ofRab8 and RablO (66% identity), the two proteins are localized to distinct cellular compartments. This approach should provide a general tool for the analyses of other members of the YPT/SEC4/rab gene family.The SEC4/YPT/Rab proteins belong to a subfamily of the Ras-like, low molecular weight GTP-binding proteins. In the budding yeast Saccharomyces cerevisiae, temperaturesensitive mutants of SEC4 accumulate secretory vesicles at the restrictive temperature, suggesting a role in post-Golgi traffic (1, 2). Likewise, yeast cells defective in YPT1 exhibit defects in early parts of the secretory pathway (3). The role of these GTP-binding proteins, as proposed by Bourne (4), is to ensure the selectivity of protein transfer by coupling the hydrolysis of GTP to the fusion events. A large number (>20) of genes belonging to this family have been identified from mammalian sources (5-14). In those cases where localization information is available, each protein appears to be localized to a distinct membrane organelle (9)(10)(11)(12)(13)(14)(15) method, we have expressed and analyzed the intracellular locations of two Rab proteins, human Rab8 and rat RablO. These two proteins were chosen because they show close sequence homologies to the yeast SEC4 protein involved in post-Golgi traffic. MATERIALS AND METHODSIsolation and Subcloning of Yeast SEC4, Human Rab8 cDNA, and Rat RablO cDNA. The S. cerevisiae temperaturesensitive sec4 mutant CHY93 (MATa leu2 ura3 sec4's) was transformed with a yeast genomic library in YCp5O vector (17) by the lithium acetate method (18). Transformants were screened for growth at 35°C and tested for cosegregation with plasmid after overnight growth in nonselective liquid medium (19). The identity of an insert retrieved from one...

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Section: Discussionmentioning

confidence: 99%

Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.

Chen

Holcomb

Moore

1993

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

139

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“…The cell suspension was homogenized in a Potter‐Elvehjen Teflon on glass homogenizer using 15 up‐and‐down strokes at 2700 r.p.m. (4°C) (Doucet et al . 1989).…”

Section: Subcellular Fractionation Of Chromaffin Cellsmentioning

confidence: 99%

Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretion

Rosé

Lejen

Casaletti

et al. 2003

Journal of Neurochemistry

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The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine b-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca 2+ and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks.

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“…Small GTP-binding proteins may also be involved in vesicle traffic (19), since GTP analogs affect intra-Golgi vesicle transport (20) and regulated secretion in neutrophils, platelets, and chromaffin cells (21)(22)(23)(24)(25). Moreover, association of noncharacterized small GTP-binding proteins with secretory vesicles has been reported (26)(27)(28)(29)(30).…”

Section: Introductionmentioning

confidence: 99%

“…27-kDa proteins, which present amino acid identities with the ras protooncogene products, might be involved in central cellular processes. In yeast, Ras proteins interact with adenylate cyclase and with inositolphospholipid turnover, suggesting a function in signal transduction (1,12).…”

Section: Introductionmentioning

confidence: 99%

Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules.

Darchen

Zahraoui

Hammel

et al. 1990

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

181

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The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells, Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine f-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTPbinding proteins, is involved in their biogenesis or in the regulated secretion process.In addition to the GTP-binding proteins involved in signal transduction, protein synthesis, or tubulin formation, a large family of small GTP-binding proteins has been discovered in eukaryotic cells (1-12). These 20-to 27-kDa proteins, which present amino acid identities with the ras protooncogene products, might be involved in central cellular processes. In yeast, Ras proteins interact with adenylate cyclase and with inositolphospholipid turnover, suggesting a function in signal transduction (1, 12). Moreover, an involvement in intracellular vesicle traffic and secretion has been shown in yeast for the Ras-like YPT1 (13,14) and SEC4 (15,16) proteins. Mutation of the SEC4 protein, which is associated with secretory vesicles and the plasma membrane, blocks a post-

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Detection of low molecular mass GTP‐binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells

Cited by 30 publications

References 29 publications

Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.

Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.

Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretion

Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules.

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