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L Johannes

Publications and source records attributed to L Johannes.

26 records · Page 2Linked to original sources
PubMed1996

Characterization of the interaction of the monomeric GTP-binding protein Rab3a with geranylgeranyl transferase II.

The monomeric GTP-binding protein Rab3a controls exocytosis in neuroendocrine and neuronal cells. Like other members of the Rab family, Rab3a is posttranslationally modified by the addition of hydrophobic geranylgeranyl groups to its C-terminus. The geranylgeranylation reaction is catalysed by the heterotrimeric geranylgeranyl transferase II. We describe the cDNA cloning of the beta-subunit of human geranylgeranyl transferase II by means of the yeast two-hybrid system. The human enzyme, which is 49% and 96% similar to yeast and rat isoforms, respectively, can complement the beta-subunit deficiency in the yeast strain ANY119. Furthermore, by means of the two-hybrid system and in vitro geranylgeranylation reactions with purified recombinant rat geranylgeranyl transferase II, we have characterized Rab3a domains implicated in the interaction with geranylgeranyl transferase II. We find that the N-terminus, the effector loop, the hypervariable region of the C-terminus, and the geranylgeranyl-acceptor cysteines have roles in this interaction. The GDP-bound form of Rab3a is the preferred substrate of geranylgeranyl transferase II.

Amino Acid Sequence↗
PubMed1996

Evidence for a functional link between Rab3 and the SNARE complex.

Rab3 is a monomeric GTP-binding protein associated with secretory vesicles which has been implicated in the control of regulated exocytosis. We have exploited Rab3 mutant proteins to investigate the function of Rab3 in the process of neurotransmitter release from Aplysia neurons. A GTPase-deficient Rab3 mutant protein was found to inhibit acetylcholine release suggesting that GTP hydrolysis by Rab3 is rate-limiting in the exocytosis process. This effect was abolished by a mutation in the effector domain, and required the association of Rab3 with membranes. In order to determine the step at which Rab3 interferes with the secretory process, tetanus and botulinum type A neurotoxins were applied to Aplysia neurons pre-injected with the GTPase-deficient Rab3 mutant protein. These neurotoxins are Zn(2+)-dependent proteases that cleave VAMP/synaptobrevin and SNAP-25, two proteins which can form a ternary complex (termed the SNARE complex) with syntaxin and have been implicated in the docking of synaptic vesicles at the plasma membrane. The onset of toxin-induced inhibition of neurotransmitter release was strongly delayed in these cells, indicating that the mutant Rab3 protein led to the accumulation of a toxin-insensitive component of release. Since tetanus and botulinum type A neurotoxins cannot attack their targets, VAMP/synaptobrevin and SNAP-25, when the latter are engaged in the SNARE complex, we propose that Rab3 modulates the activity of the fusion machinery by controlling the formation or the stability of the SNARE complex.

Acetylcholine↗
PubMed1994

The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells.

There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic along biosynthetic and endocytotic pathways in eukaryotic cells. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. We report here that overexpression in PC12 cells of Rab3a mutant proteins defective in either GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. Finally, the electrophysiological approach showed that bovine chromaffin cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. In contrast, control cells showed a phenomenon of desensitization. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis from occurring unless secretion is triggered. Furthermore, it is proposed that Rab3a is involved in adaptive processes such as response habituation.

Adrenal Cortex↗
PubMed1994

Rab3 proteins: key players in the control of exocytosis.

Although some mechanistic aspects of exocytosis, such as fusion events, have been well documented by the technique of time-resolved membrane-capacitance measurement, it was only recently that new insights into the molecular mechanisms involved in the traffic of secretory vesicles were provided by the convergence of different lines of research. In this review Lledo et al. present some of the recent findings concerning small GTPases of the Rab3 subfamily which regulate hormone release, triggered by entry of Ca2+, in endocrine and neuroendocrine cells. In view of these new results, Rab proteins might be considered as candidates for inhibition or stimulation of specific steps involved in vesicle traffic.

Animals↗
PubMed1993

Characterization of two novel single-stranded DNA-specific autonomously replicating sequence-binding proteins from Saccharomyces cerevisiae, one of which is adenylosuccinate synthetase.

We report here the identification and characterization of two novel proteis from Saccharomyces cerevisiae that bind to the T-rich strand of the core consensus autonomously replicating sequence (ARS) in a highly specific manner. The two proteins, 40 and 45 kDa in size, can be distinguished by multiple criteria from each other and from the 65-kDa ssArS-T-binding protein identified recently in our laboratory (Schmidt, A. M. A., Herterich, S. U., and Krauss, G. (1991) EMBO J. 10, 981-985). The specificity of binding is inferred from gel shift and nuclease-footprinting experiments using single-stranded probes containing the core consensus ARS. With a 321-nucleotide single-stranded ARS1 fragment, specific protection of the A and B1 domain against DNase I digestion is observed. Partial amino acid sequencing and enzymatic assays identify the 45-kDa protein as adenylosuccinate synthetase, an enzyme necessary for the de novo synthesis of adenylate.

Adenylosuccinate Synthase↗
PubMed1993

Switch in antiviral specificity of a GTPase upon translocation from the cytoplasm to the nucleus.

The Mx2 protein of rats is a cytoplasmic GTPase that protects cells against vesicular stomatitis virus but not against influenza virus. Since vesicular stomatitis virus replicates in the cytoplasm and influenza virus replicates in the nucleus, it was possible that the antiviral specificity of rat Mx2 protein was determined solely by the protein's subcellular localization. Here, we found that, indeed, rat Mx2 protein lost its anti-vesicular stomatitis virus activity and gained anti-influenza virus activity when it was directed to the nucleus by way of a foreign nuclear-transport signal appended to its amino terminus. These data show that rat Mx2 protein possesses an antiviral activity that is revealed only when the protein is shuttled to the nucleus.

3T3 Cells↗
PubMed1993

[Calcium-dependent regulated secretion is controlled by GTPase Rab3 in neuroendocrine cells].

Changes in intracellular free calcium concentration ([Ca2+]i) play a fundamental role in the regulation of hormone secretion by endocrine and neuronal cells. The increase in [Ca2+]i is one of the principle events in stimulus-secretion coupling. Secretory phenomena are subjected to modulation via a large number of intracellular factors which can act on vesicular transport, exocytosis or endocytosis. Among these factors are proteins able to bind and hydrolyse the nucleotide GTP belonging to the ras superfamily of small GTPases (or superfamily of small G proteins). GTPases of the Sec4/Ypt1/Rab family have been implicated in the regulation of intracellular vesicle traffic. A role for Rab3 in regulating secretion was initially proposed because of its specific expression in the nervous and endocrine systems, and its association with secretory vesicles. The function of Rab3 has been studied in two types of neuroendocrine cells: the anterior pituitary lactotroph cells and the adrenal medulla chromaffin cells. Two techniques were combined: intracellular injection of antisense oligonucleotides and recombinant proteins, followed by membrane capacitance measurements. The inhibition of Rab3b synthesis in lactotroph cells by antisense oligonucleotides provokes an inhibition of exocytosis. On the other hand, injection of antisense oligonucleotides directed against rab3a into chromaffin cells led to a stimulation of the secretory response to successive depolarizations of the cell membrane. These results indicate that Rab3 proteins control the calcium-dependent secretory process in neuroendocrine cells. In lactotroph cells Rab3b seems to play a stimulatory role, whereas Rab3a acts as a negative regulator of catecholamine release in chromaffin cells.

Animals↗