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The Rho GTPase promotes proliferation and cytoskeletal rearrangements in mammalian cells. To understand the regulation of Rho, it is important to characterize guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of GDP and subsequent binding of GTP. Using Rho as an affinity ligand, we have isolated a 115-kDa protein (p115-RhoGEF) that binds specifically to the nucleotide-depleted state. A fulllength cDNA encoding p115-RhoGEF was isolated, and its protein product, which exhibited sequence homology to Dbl and Lbc, catalyzed the exchange of GDP for GTP specifically on Rho and not on the Rac, Cdc42, or Ras GTPases. p115-RhoGEF is capable of regulating cell proliferation, as determined by its ability to induce the transformation of NIH 3T3 cells. Northern and Western analysis suggests that p115-RhoGEF is ubiquitously expressed. These results indicate that p115-RhoGEF may be a general regulator of Rho and its associated cellular phenotypes.

The small GTPase Rac has been implicated in a wide range of cellular processes, including the organization of the actin cytoskeleton, transcriptional control and endocytic vesicle trafficking [1-3]. The signaling components that mediate these functions downstream of Rac largely remain to be identified. In this study, we have identified synaptojanin 2, a polyphosphoinositide phosphatase as a novel Rac1 effector. Synaptojanin 2 directly and specifically interacts with Rac1 in a GTP-dependent manner. Expression of constitutively active Rac1 caused the translocation of synaptojanin 2 from the cytoplasm to the plasma membrane. Both activated Rac1 and a membrane-targeted version of synaptojanin 2 inhibited endocytosis of the epidermal growth factor (EGF) and transferrin receptors, a process that is known to be dependent on polyphosphoinositide lipids. Endocytosis of growth factor receptors is thought to play an important role in the regulation of cell proliferation. Thus, these results suggest that synaptojanin 2 may mediate the inhibitory effect of Rac1 on endocytosis and could contribute to Rac1-mediated control of cell growth.

A strain of Saccharomyces cerevisiae capable of simultaneous hydrolysis and fermentation of highly polymerized starch oligosaccharides was constructed. The Aspergillus awamori glucoamylase enzyme, form GAI, was expressed in Saccharomyces cerevisiae by means of the promoter and termination regions from a yeast enolase gene. Yeast transformed with plasmids containing an intron-free recombinant glucoamylase gene efficiently secreted glucoamylase into the medium, permitting growth of the transformants on starch as the sole carbon source. The natural leader sequence of the precursor of glucoamylase (preglucoamylase) was processed correctly by yeast, and the secreted enzyme was glycosylated through both N- and O-linkages at levels comparable to the native Aspergillus enzyme. The data provide evidence for the utility of yeast as an organism for the production, glycosylation, and secretion of heterologous proteins.