A Zea mays GTP-binding protein of the ARF family complements an Escherichia coli mutant with a temperature-sensitive malonyl-coenzyme A:acyl carrier protein transacylase

The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the deltabem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the deltabem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.

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Pertussis toxin substrate is a guanosine 5′-[β-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein

Biochemical Journal ◽

10.1042/bj2320191 ◽

1985 ◽

Vol 232 (1) ◽

pp. 191-197 ◽

Cited By ~ 23

Author(s):

S K Wong ◽

B R Martin ◽

A M Tolkovsky

Keyword(s):

Pertussis Toxin ◽

Binding Protein ◽

Alkylating Agents ◽

Guanine Nucleotides ◽

Temperature Sensitive ◽

Gtp Binding Protein ◽

Adp Ribosylation ◽

Gtp Binding ◽

Rat Brain And Liver ◽

Liver Membranes

We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5′-[γ-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5′-[β-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guanosine 5′-[β, γ-imido]triphosphate (p[NH]ppG). The order of labelling intensity was GDP[S] greater than GTP greater than no addition greater than GTP[S] = p [NH]ppG. Mg2+ increased labelling by CT, but decreased labelling by IAP. In addition, Mg2+ potentiated the effects of the guanine nucleotides, increasing the inhibitory effects of GTP[S] and the activatory effects of GDP[S] or GTP. Preincubating liver membranes at 30 degrees C in the presence of 10 mm-MgCl2 inhibited labelling by IAP irreversibly. Pretreatment of liver membranes with 4.95 mM-N-ethylmaleimide decreased labelling by CT by approximately 15%, but almost completely blocked labelling by IAP. These results suggest that the undissociated, GDP-bound, conformation of Ni, the inhibitory GTP-binding protein of adenylate cyclase, is the preferred substrate for ADP-ribosylation by IAP. This conformation, which is prevalent in native membranes, is sensitive to temperature, Mg2+ ions and alkylating agents such as N-ethylmaleimide. At 30 degrees C, Mg2+ may cause dissociation and denaturation of Ni in native membranes.

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