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Yeast cells of Candida dbicans which had been attached to polylysine-coated microscope slides were induced to form buds or germ tubes in the presence of external electrical fields. The sites of budding and germ tube formation and the growth of germ tubes and hyphal branches were polarized preferentially towards the cathode. Buds were not converted to pseudohyphae or germ tubes by the field and the field had no effect on the positioning of nuclei or septa in the yeast cell or germ tube. Buds were less polarized than germ tubes at any given applied voltage. The polarization of buds reached a peak at an electrical field of 12 mV per cell. Polarization of germ tubes was biphasic, incri5adihgrapidly with %cre*&hig field strengths up to 5 mV per cell, and then more slowly in stronger fields. An electrical field was only required for a fraction of the time taken for germ tubes to start to form, so cells retained a memory of experiencing an electrical field which influenced the selection of sites of evagination. Increasing the electrical field delayed the time of germ tube evagination and inhibited the rate of germ tube extension. Unlike previous findings with other filamentous fungi, germ tubes grew unidirectionally towards the cathode for extended periods and did not deviate to a perpendicular orientation. This result suggests that the septa1 pore of the filamentous form may have high electrical resistance and would act as an effective barrier to solute transport between intercalary compartments.

The yeast TRP3 gene encodes a bifunctional protein with anthranilate synthase I1 and indoleglycerol-phosphate synthase activities. Replacing ten consecutive non-preferred codons in the indoleglycerol-phosphate synthase region of the TRP3 gene with synonymous preferred codons (to create the TRP3pr gene ; translational pause replaced) causes a 1 .S-fold reduction in relative indoleglycerolphosphate synthase activity [Crombie, T., Swaffield, J. C. & Brown, A. J. P. (1992) J. Mol. Biol. 228,121. Here, we report that both the anthranilate synthase I1 and indoleglycerol-phosphate synthase domains are affected to similar extents when the translational pause is removed. Also, structural modelling of the yeast indoleglycerol-phosphate synthase domain against the X-ray crystal structure of indoleglycerol-phosphate synthase from Escherichia coli indicates that the translational pause lies in a region of structural divergence between similar structures. To probe the role of cytoplasmic heat-shock protein 70 (Hsp7O) chaperones in Trp3 protein folding, anthranilate synthase and indoleglycerol-phosphate synthase activities were measured in ssa and ssb mutants. Neither indoleglycerol-phosphate synthase nor anthranilate synthase were affected significantly in the ssb mutant. However, depletion of Hsp7O proteins encoded by the SSA genes led to decreased anthranilate synthase and indoleglycerol-phosphate synthase activities from the TRP3 gene, suggesting that both domains depend to some extent upon the SSA chaperone family. The data are consistent with roles for both the translational pause and Ssa chaperones in Trp3 protein folding in vivo.Detailed analyses of protein refolding in vitro have provided considerable insights into the means by which polypeptide chains attain their native three-dimensional conformation (for reviews, see Creighton, 1990;Fischer and Schmid, 1990;Kim and Baldwin, 1990;Christensen and Pain, 1991 ;Scholtz and Baldwin, 1992;Matthews, 1993). These studies have highlighted the importance of the spatial organization of amino acids within the polypeptide chain in the folding process. However, additional factors influence protein folding in vivo, including chaperones, protein disulphide isomerases and peptidyl prolyl cis-trans isomerases (for reviews, see Freedman, 1989;Fischer and Schmid, 1990;Gething and Sambrook, 1992;Hartl et al., 1992;Craig et al., 1993;Hendrick and Hartl, 1993). In addition, the temporal separation of amino acids during the synthesis of the nascent polypeptide chain might be significant for protein folding in vivo (Phillips, 1966;Baldwin, 1975;Goldberg, 1985).Previously, we proposed that distinct translational pauses have evolved in some mRNAs to promote protein folding in vivo by temporally separating the folding of specific regions of their polypeptide chains (Purvis et al., 1987). The hypothesis was based upon the following observations. (a) Nascent polypeptides start to fold as they are being synthesized (Bergman and Kuehl, 1979a;Bergman and Kuehl, 1979b;Peters and Davidson, 1982). (...

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