Genetic Analysis of the Saccharomyces Cerevisiae Rho3 Gene, Encoding a Rho-Type Small Gtpase, Provides Evidence for a Role in Bud Formation

RHO3 encodes a Rho-type small GTPase of the yeast Saccharomyces cerevisiae. We isolated temperature-sensitive alleles and a dominant active allele of RHO3. Ts(-) rho3 cells lost cell polarity during bud formation and grew more isotropically than wild-type cells at nonpermissive temperatures. In contrast, cells carrying a dominant active mutant RHO3 displayed cold sensitivity, and the cells became elongated and bent, often at the position where actin patches were concentrated. These phenotypes of the rho3 mutants strongly suggest that RHO3 is involved in directing the growing points during bud formation. In addition, we found that SRO6, previously isolated as a multicopy suppressor of rho3, is the same as SEC4. The sec4-2 mutation was synthetic lethal with temperature-sensitive rho3 mutations and suppressed the cold sensitivity caused by a dominant active mutant RHO3. The genetic interactions between RHO3 and SEC4, taken together with the fact that the Rab-type GTPase Sec4p is required to fuse secretory vesicles together with plasma membrane for exocytosis, support a model in which the Rho3p pathway modulates morphogenesis during bud growth via directing organization of the actin cytoskeleton and the position of the secretory machinery for exocytosis.     

A novel rho promoter::Tn10 mutation suppresses and ftsQ1(Ts) missense mutation in an essential Escherichia coli cell division gene by a mechanism not involving polarity suppression.

An extragenic suppressor of the Escherichia coli cell division gene ftsQ1(Ts) was isolated. The suppressor is a Tn10 insertion into the -35 promoter consensus sequence of the rho gene, designated rho promoter::Tn10. The ftsQ1(Ts) mutation was also suppressed by the rho-4 mutant allele. The rho promoter::Tn10 strain does not exhibit rho mutant polarity suppressor phenotypes. In addition, overexpression of the ftsQ1(Ts) mutation does not reverse temperature sensitivity. Furthermore, DNA sequence analysis of the ftsQ1(Ts) allele revealed that the salt-remediable, temperature-sensitive phenotype arose from a single missense mutation. The most striking phenotype of the rho promoter::Tn10 mutant strain is an increase in the level of negative supercoiling. On the basis of these observations, we conclude that the ftsQ1(Ts) mutation may be suppressed by a change in supercoiling.     

An RNA-dependent ATPase from Chlamydomonas reinhardII

An RNA-dependent ATPase has been isolated from extracts of Chlamydomonas reinhardii. The enzyme catalyzes the hydrolysis of ATP, dATP, CTP and dCTP to the corresponding nucleoside diphosphate and Pi in the presence of Mg2+ or Mn2+ and an RNA cofactor. In 1 mM MgCl2 it displays the greatest activity with poly(A), poly(I) and poly(U); and somewhat lower activity with poly(C) and T7 RNA. Although the enzyme is active with single-stranded DNA, all the single-stranded RNAs tested were significantly more effective cofactors than any of the single or double-stranded DNAs tested. A comparison of this ATPase with other RNA-dependent ATPases indicates that is has more in common with the ATPase isolated from the nuclei of animal cells than with the RNA synthesis termination protein rho, the major RNA-dependent ATPase from Escherichia coli. Although chloroplasts of C. reinhardii are known to contain many bacterial-like gene expression components, the presence of an enzyme with close homology to the E. coli rho protein was not detected.     

Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae. Decoupling of intracellular function from ATPase activity and hexamerization

The AAA protein Drg1 from yeast was affinity-purified, and its ATPase activity and hexamerization properties were analyzed. The same parameters were also determined for several mutant proteins and compared in light of the growth characteristics of the corresponding cells. The protein from a thermosensitive mutant exhibited reduced ATPase activity and hexamerization. These defects were not reversed by an intragenic suppressor mutation, although this allele supported growth at the nonpermissive temperature. A different set of mutants was generated by site-specific mutagenesis intended to adjust the Walker A box of the D2 domain of Drg1p to that of the D1 domain. A S562G exchange in D2 produced a nonfunctional protein that did not hexamerize but showed above-normal ATPase activity. The C561T mutant protein, on the other hand, was functional but hexamerized less readily and had reduced ATPase activity. In contrast, the C561T/S562G protein hexamerized less than wild type but had much higher ATPase activity. We distinguished strong and weak ATP-binding sites in the wild type protein but two weak sites in the C561T/S562G protein, indicating that the stronger site resides in D2. These observations are discussed in terms of the inter-relationship of ATPase activity per se, oligomeric status, and intracellular function for AAA proteins.     

Mutant analysis reveals a specific requirement for protein P30 in Mycoplasma pneumoniae gliding motility

The cell-wall-less prokaryote Mycoplasma pneumoniae, long considered among the smallest and simplest cells capable of self-replication, has a distinct cellular polarity characterized by the presence of a differentiated terminal organelle which functions in adherence to human respiratory epithelium, gliding motility, and cell division. Characterization of hemadsorption (HA)-negative mutants has resulted in identification of several terminal organelle proteins, including P30, the loss of which results in developmental defects and decreased adherence to host cells, but their impact on M. pneumoniae gliding has not been investigated. Here we examined the contribution of P30 to gliding motility on the basis of satellite growth and cell gliding velocity and frequency. M. pneumoniae HA mutant II-3 lacking P30 was nonmotile, but HA mutant II-7 producing a truncated P30 was motile, albeit at a velocity 50-fold less than that of the wild type. HA-positive revertant II-3R producing an altered P30 was unexpectedly not fully wild type with respect to gliding. Complementation of mutant II-3 with recombinant wild-type and mutant alleles confirmed the correlation between gliding defect and loss or alteration in P30. Surprisingly, fusion of yellow fluorescent protein to the C terminus of P30 had little impact on cell gliding velocity and significantly enhanced HA. Finally, while quantitative examination of HA revealed clear distinctions among these mutant strains, gliding defects did not correlate strictly with the HA phenotype, and all strains attached to glass at wild-type levels. Taken together, these findings suggest a role for P30 in gliding motility that is distinct from its requirement in adherence.