Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1 cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.The yeast RNA polymerase III (Pol III) transcription system is well characterized. Small untranslated RNAs with essential housekeeping functions, such as tRNAs, 5S rRNA, or the U6 small nuclear RNA (snRNA) that is required for mRNA splicing, are synthesized by Pol III with the help of two general auxiliary factors, TFIIIC and TFIIIB. The large TFIIIC factor (six subunits) binds to the DNA promoter elements and assembles the initiation factor TFIIIB (three components) upstream of the start site. Once TFIIIB is in position, it recruits the Pol III enzyme (17 subunits) and directs accurate and multiple rounds of transcription. All of the polypeptide components of the Pol III apparatus (ϳ1,500 kDa) have been characterized and found to be essential for cell viability (8,23). The identification of the components of the Pol III system has facilitated the description of a cascade of protein-protein interactions that leads to the recruitment of the Pol III enzyme (reviewed in reference 55).Detailed knowledge of the yeast Pol III transcription system contrasts with the limited information available on the control of class III gene expression in yeast. Cellular tRNA levels respond to cell growth rate (48,49), to a nutritional upshift (27,48) or to nitrogen starvation (36) but only modestly to amino acid starvation (41). Finally, Pol III transcription is repressed in secretion-defective cells (30). Although the mechanism of repression is not clear, it does involve activation of the cell integrity pathway (30). The effect of growth conditions on Pol III transcription is well mimicked in vitro with whole-cell extracts (11, 39). tRNA synthesis is downregulated in dense cell cultures approaching stationary phase, a result due essentially to reduced TFIIIB activity. The TFIIIB component Brf/ TFIIIB70 was found to be the limiting factor in extracts from such cells (39). However, the occupancy of the TFIIIB binding site on the SUP53 gene encoding tRNA Leu does not decrease in stationary-phase cells. Rather, in vivo footprinting data suggest reduced p...

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.

Gametogenesis in Chlamydomonas reinhardtii has been studied in mating-type plus cells utilizing several different culture conditions, all of which are shown to depend on the depletion of nitrogen from the medium, and the fine structure of gametes prepared under these conditions has been compared by using thin sections of fixed material. We document alterations in ribosome levels, in chromatin morphology, in starch levels, in the organization of chloroplast membranes, and in the appearance of nuclear envelope and endoplasmic reticulum membranes during gametogenesis. We also note the acquisition of two new organelles: a mating structure (Friedman, L., A. L. Colwin, and L. H. Colwin. 1968. J. Cell Sci. 3:115-128; Goodenough, U. W., and R. L. Weiss. 1975. J. Cell Biol. 67:623-637), and Golgi-derived vesicles containing a homogeneous material. We chart the time course of these morphological changes during synchronous gametogenesis. We note that many of these changes may represent adjustments to nitrogen starvation rather than direct features of gametic differentiation, and we also document that cells can differentiate so that they survive conditions of nitrogen starvation for many weeks after they become gametes. We conclude that metabolic alterations, the acquisition of mating ability, and the preparation for long-term survival are all elicited in this organism by nitrogen withdrawal, and we discuss how the various structural alterations observed in this study may relate to these three interrelated avenues of cellular differentiation.When the unicellular flagellate Chlarnvdomonas reinhardtii is induced to differentiate from a vegetative cell into a gamete, a number of changes occur in its biochemistry, morphology, and behavior that have been described by earlier investigators. The differentiation period itself, which can be as short as 10-12 h in synchronized cultures (16), requires both RNA and protein synthesis (14) and is marked by an extensive breakdown and resynthesis of nucleic acids (20) and an alteration in the activity of several enzymes (15). Synchronous differentiation normally concludes with a mitotic division, and the gametic cells that emerge from this mitosis are able to mate with 100% efficiency (3). Mating is induced by mixing gametes of mating-type plus (rot +) with those of rot-. The cells first agglutinate by the tips of their flagella; there follow a shedding of cell walls (4) and a cell fusion between pairs of mt + and rot-gametes to form quadriflagellated zygotes (18).We initiated a fine-structural study of gametogenesis in C. reinhardtii as a prelude to analyzing