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SummarySmall GTP-binding proteins of the highly conserved Rho family act as molecular switches regulating cell signalling, cytoskeletal organization and vesicle trafficking in eukaryotic cells. Here we show that in the dimorphic plant pathogenic fungus Ustilago maydis deletion of either cdc42 or rac1 results in loss of virulence but does not interfere with viability. Cells deleted for cdc42 display a cell separation defect during budding. We have previously shown that the Rhospecific guanine nucleotide exchange factor (GEF) Don1 is required for cell separation in U. maydis . Expression of constitutive active Cdc42 rescues the phenotype of don1 mutant cells indicating that Don1 triggers cell separation by activating Cdc42. Deletion of rac1 affects cellular morphology and interferes with hyphal growth, whereas overexpression of wild-type Rac1 induces filament formation in haploid cells. This indicates that Rac1 is both necessary and sufficient for the dimorphic switch from budding to hyphal growth. Cdc42 and Rac1 share at least one common essential function because depletion of both Rac1 and Cdc42 is lethal. Expression of constitutively active Rac1 Q61L is lethal and results in swollen cells with a large vacuole. The morphological phenotype, but not lethality is suppressed in cla4 mutant cells suggesting that the PAK family kinase Cla4 acts as a downstream effector of Rac1.

The transcription/DNA repair factor TFIIH may be resolved into at least two subcomplexes: the core TFIIH and the cdk-activating kinase (CAK) complex. The CAK complex, which is also found free in the cell, is composed of cdk7, cyclin H, and MAT1. In the present work, we found that the C terminus of MAT1 binds to the cdk7⅐cyclin H complex and activates the cdk7 kinase activity. The median portion of MAT1, which contains a coiled-coil motif, allows the binding of CAK to the TFIIH core through interactions with both XPD and XPB helicases. Furthermore, using recombinant TFIIH complexes, it is demonstrated that the N-terminal RING finger domain of MAT1 is crucial for transcription activation and participates to the phosphorylation of the C-terminal domain of the largest subunit of the RNA polymerase II. Cyclin-dependent kinases (cdk)1 have a central role in the coordination of the eukaryotic cell cycle and participate in the integration of diverse growth regulatory signals. Some members of this protein kinase family are involved not only in cell cycle control but also in other mechanisms that govern cell life, notably transcription. Cdk activities are regulated by several different processes including the binding of an activating cyclin subunit, their phosphorylation, and the association of cdk inhibitors or other stimulatory factors (1). One of them, cdk7, was originally identified as the catalytic subunit of the cdk-activating kinase (CAK) complex that is able to phosphorylate, and thereby activate, several cdks (2). Besides cdk7, the CAK complex is composed of the regulatory subunit cyclin H (3, 4) and a third partner, MAT1, defined as a stabilizing factor (5, 6).Yeast genetic as well as biochemical studies demonstrated that MAT1 is also, together with cyclin H and cdk7, part of the general transcription factor TFIIH (7,8). TFIIH is composed of nine subunits and can be functionally divided into several subcomplexes such as the core TFIIH and the CAK complex (9, 10). TFIIH plays a role not only in transcription but also in DNA repair (11). Although TFIIH factor is absolutely required in nucleotide excision repair (11), the role of the CAK subcomplex in this reaction is not clear (12).Cdk7 phosphorylates several basal transcription factors (13-15) as well as the C-terminal domain (CTD) of the largest subunit of the RNA polymerase II (RNA pol II) (16). However, cdk7 is not the only kinase responsible for CTD phosphorylation. Indeed, the cdk8⅐cyclin C complex, which is found associated with the holoenzyme transcription complex, phosphorylates the CTD in vitro (17). Furthermore, the Srb10⅐Srb11 complex (the yeast counterpart of cdk8⅐cyclin C) phosphorylates the CTD in vivo prior to the formation of the preinitiation complex (PIC), resulting in an inhibition of the transcription reaction (18). Also, during human immunodeficiency virus infection, the CTD kinase activity of the cdk9⅐cyclin T complex, characterized as a component of the elongation factor pTEFb (for positive transcription elongation factor b), is required fo...

SummaryThe phytopathogenic basidiomycete Ustilago maydis displays a dimorphic switch between budding growth of haploid cells and filamentous growth of the dikaryon. In a screen for mutants affected in morphogenesis and cytokinesis, we identified the serine/threonine protein kinase Cla4, a member of the family of p21-activated kinases (PAKs). Cells, in which cla4 has been deleted, are viable but they are unable to bud properly. Instead, cla4 mutant cells grow as branched septate hyphae and divide by contraction and fission at septal cross walls. Delocalized deposition of chitinous cell wall material along the cell surface is observed in cla4 mutant cells. Deletion of the Cdc42/ Rac1 interaction domain (CRIB) results in a constitutive active Cla4 kinase, whose expression is lethal for the cell. cla4 mutant cells are unable to induce pathogenic development in plants and to display filamentous growth in a mating reaction, although they are still able to secrete pheromone and to undergo cell fusion with wild-type cells. We propose that Cla4 is involved in the regulation of cell polarity during budding and filamentation.

Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3 and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis.Morphological switching is a unique attribute of all dimorphic fungi, which alternate between budding and filamentous growth. In some cases, as with mating, this is a prerequisite for genetic diversity for this subfamily of fungi. In addition, many dimorphic fungal pathogens rely on this ability in order to effectively invade their host. In general, the transition between these alternate life forms means a complete turnover of cellular and proteomic components, which often involves cell cycle arrest and/or cytoskeletal rearrangement. Although the cellular proteomes associated with these two processes share many components, there are both temporal and spatial regulations that are manifested during the transitional phase (4).Temporal-spatial regulation of the proteome during the dimorphic transition requires cooperation and synchronized communication among different regulatory pathways. Two highly intricate, yet well-established, signaling cascades that regulate fungal morphogenesis are the mitogen-activated protein kinase (MAPK) (34, 46) and protein kinase A pathways (11). These signaling cascades detect and perpetuate extracellular stimuli, e.g., pheromones and nutrients, which lead to phase transitions in dimorphic fungi. Although the mechanisms are not as fully understood, members of two highly conserved families of proteins, Rho/Rac GTPases and 14-3-3 proteins, have also been shown to control filamentation. Constituents of the Rho/Rac protein family have been shown to regulate actin organization (26,35,36), cytokinesis (3, 49, 52), cell integrity (42, 56), pathogenicity (29), signal transduction (22,44,56), and cell migration (8). Their activity is dependent...

HeLa cell extracts contain significant amounts of terminal uridylyl transferase (TUTase) activity. In a template-independent reaction with labeled UTP, these enzymes are capable of modifying a broad spectrum of cellular RNA molecules in vitro . However, fractionation of cell extracts by gel filtration clearly separated two independent activities. In addition to a non-specific enzyme, an additional terminal uridylyl transferase has been identified that is highly specific for cellular and in vitro synthesized U6 small nuclear RNA (snRNA) molecules. This novel TUTase enzyme was also able to select as an efficient substrate U6 snRNA species from higher eucaryotes. In contrast, no labeling was detectable with purified fission yeast RNA. Using synthetic RNAs containing different amounts of transcribed 3'-end UMP residues, high resolution gel electrophoresis revealed that U6 snRNA species with three terminal U nucleotides served as the optimal substrate for the transferase reaction. The 3'-end modification of the optimal synthetic substrate was identical to that observed with endogenous U6 snRNA isolated from HeLa cells. Therefore, we conclude that the specific addition of UMP residues to 3'-recessed U6 snRNA molecules reflects a recycling process, ensuring the functional regeneration for pre-mRNA splicing of this snRNA.

To understand the role of the various components of TFIIH, a DNA repair/transcription factor, a yeast fourhybrid system was designed. When the ternary Cdkactivating kinase (CAK) complex composed of Cdk7, cyclin H, and MAT1 was used as bait, the xeroderma pigmentosum (XP) D helicase of transcription factor IIH (TFIIH), among other proteins, was identified as an interacting partner. Deletion mutant analyses demonstrated that the coiled-coil and the hydrophobic domains of MAT1 interlink the CAK complex directly with the N-terminal domain of XPD. Using immunoprecipitates from cells coinfected with baculoviruses, we further validated the bridging function of XPD, which anchors CAK to the core TFIIH. In addition we show that upon interaction with MAT1, CAK inhibits the helicase activity of XPD. This inhibition is overcome upon binding to p44, a subunit of the core TFIIH. It is not surprising that under these conditions some XPD mutations affect interactions not only with p44, but also with MAT1, thus preventing either the CAK inhibitory function within CAK⅐XPD and/or the role of CAK within TFIIH and, consequently, explaining the variety of the XP phenotypes.DNA replication, transcription, and translation are regulated through the ordered formation of large protein complexes, the activity of which is often modulated by protein-protein interactions as well as protein modifications such as phosphorylation, acetylation, methylation, etc. Several large complexes such as RNA polymerases (1), TFIID 1 (2), the transcription/ DNA repair factor TFIIH (3, 4), or the mediator complexes (5) are involved in transcription initiation. To ensure that these complexes work properly, it seems likely that they might be regulated by other cellular components. These regulations may involve subtle and weak interactions between partners that are hardly detectable with classical methods such as the yeast two-hybrid system or immunoprecipitation using one partner as bait. To further understand the internal interactions within these large complexes and/or to identify the externally interacting partner(s), we theorize that the bait should be a multiprotein complex. In such a case, the target protein would recognize a specific structure displayed by the subunits of the complex used as bait. This multipoint surface will likely increase and stabilize the interaction between the target protein to be characterized and the bait.The transcription/DNA repair factor TFIIH is a complex enzyme composed of nine subunits, three of which (XPB, XPD, and Cdk7) have identified enzymatic activities (6). Once recruited either by the stable transcription preinitiation complex or by the damage recognition complex, the XPB and XPD helicases of TFIIH induce the opening of the DNA around the transcription start site and the lesion, respectively (6 -9). Interestingly, mutations in these two proteins affect some of the enzymatic activities of TFIIH and give rise to three autosomal recessive genetic disorders, Xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiody...

In the dimorphic fungus Ustilago maydis the Rho-family GTP-binding protein Cdc42 and the Ste20-like kinase Don3 are both essential for triggering cell separation during cytokinesis. Since Don3 does not contain a Cdc42/Rac interaction and binding domain (CRIB), it is unclear how Cdc42 and Don3 cooperate in the regulation of cytokinesis. To analyse the regulatory network we generated an analogue-sensitive Don3 variant (Don3-as) that allows specific inhibition in vivo. The engineered kinase Don3M157A is fully active in vivo and can be specifically inhibited by low concentrations of the ATP-analogue NA-PP1. Inhibition of the Don3-as kinase activity immediately blocked cell separation resulting in the formation of clusters of nonseparated cells. Covalent labelling of cell wall proteins showed that, upon release of inhibition, cytokinesis was resumed instantaneously in all cells. By sequentially activating Don3 and Cdc42 we were able to demonstrate that both proteins act independently of each other and that Don3 activity precedes that of Cdc42. We provide evidence that Don3 and Cdc42 are crucial for the assembly of a contractile actomyosin ring, which is a prerequisite for secondary septum formation. We propose, that Don3 is involved in establishing a landmark, at which the Cdc42-dependent actomyosin ring formation will occur.