Browsing by Author "Karnam, Harish Kumar"

Filter results by typing the first few letters
Now showing 1 - 1 of 1
  • Thumbnail Image
    ItemOpen Access
    The novel proteasomal substrate Far10 contributes to control of mitotic exit in yeast
    (2005) Karnam, Harish Kumar; Hilt, Wolfgang (Priv. Doz. Dr. )
    Ubiquitin-Proteasome System (UPS) mediated proteolysis of an array of cellular proteins plays an important role in many basic physiological processes. Among these are control of cell cycle and division, differentiation and development, response to stress, transcriptional regulation, circadian rhythms, regulation of the immune and inflammatory responses, and biogenesis of organelles. Some of the well-known substrates of this system are cell cycle regulators such as cyclins, cyclin dependent kinase inhibitors, and proteins involved in sister chromatid separation, tumor suppressors, as well as transcriptional activators and their inhibitors [Glickman, 2002; Hilt, 2004; Wolf, D.H, 2004]. Due to these facts, identification and characterization of new substrates of the ubiquitin-proteasome system is important to reveal its cellular functions. For this purpose a high expression lethality [HEL] screen had been developed [Ledig, 1996; Velten, 1996, Velten, 2000]. This screen was based on the hypothesis that overexpression of a protein whose degradation by the ubiquitin-proteasome system is required for viability or growth, will cause a strong growth defect in cells where proteasome function is impaired, as for instance in pre1-1 pre4-1 mutants. An unknown protein originally designated as Hel48 now commonly termed as Far10 was identified, [Velten, 2000; Kemp and Sprague, Jr., 2003]. In this work cycloheximide chase experiments were undertaken to prove that Far10 is a novel substrate of the proteasome. Far10 expressed from its endogenous promoter on the chromosome either as N-terminally 19Myc tagged or as C-terminally 3Ha-tagged version was rapidly degraded in wild type cells and stabilized in pre1-1 pre4-1 proteosome mutants. Based on the ability of HA-tagged Far10 to cause lethality it was concluded that the tagged version of this protein is functional. Therefore the degradation rates seen with different tagged versions are supposed to be as wild type. The identical behavior of N-terminally and C-terminally tagged Far10 strongly support this idea. Regulatory proteolysis is an important mechanism for major cell cycle transitions such as the initiation of DNA replication, separation of sister chromatids and exit from mitosis [Jan-Michael Peters, 1998; Hilt, 2004]. APC, an ubiquitin-protein ligase, consisting of 12 known subunits in Saccharomyces cerevisiae is essential for ubiquitin-dependent proteolysis during mitosis [Harper et al., 2002; Jan-Michael Peters, 2002]. It requires two substrate specific co-activators: Cdc20 and Cdh1/Hct1. Substrates of APCCdc20 complex include non-cyclins such as Pds1 [Cohen-Fix et al., 1996; Michaelis et al., 1997; Ciosk et al., 1998; Nasmyth, 1999] and cyclins such as Clb2 and Clb5 [Bäumer et al., 2000; Wäsch, 2002; Irniger, 2002; Cross, 2003]. APCCdh1 complex initiates degradation of the mitotic cyclin Clb2 in telophase and also mediates proteolysis of other proteins such as the spindle-associated protein Ase1, Cdc20 and the polo-like kinase Cdc5 [Schwab et al., 1997; Visintin et al., 1997; Shirayama et al, 1998]. Thus, Cdc20 and Cdh1 ensure that different target proteins of the APC are degraded in a proper temporal order during mitosis. The participation of the anaphase-promoting complex and its co-activators in the degradation of Far10 was demonstrated by the observation of synthetic dosage effects in cdc23-1, cdc20-1 and hct1-?1 mutants. Cycloheximide decay analysis of 19Myc tagged Far10 in cdc23-1 APC mutants as well as cdc20-1 and proteasome mutants uncovered a clear proteolytic stabilization of N(myc)19Far10. On the contrary, a deletion of HCT1 had no effect on the degradation of Far10. These results confirm that Far10 is a genuine substrate of the APC and requires the specificity factor Cdc20 for its degradation. In addition to this, analysis of in-vivo ubiquitination experiments of Far10(HA)3 in wild type (WCG4) and pre1-1 pre4-1 proteasome mutants revealed that the polyubiquitinated forms of Far10(HA)3 accumulate in the pre1-1 pre4-1 proteasome mutants. Substrates of APC and Cdc20 in particular identified till date have a nine amino acid conserved motif called the destruction [D] box which has a consensus sequence: RXXLXXVXN/D/E. Far10 being a substrate of APCCdc20 has a nine amino-acid sequence similar to the D box motif, 340RRKLSGKYE348 residing in the C-terminal region. To check the relevance of this motif in the degradation of Far10, site directed mutagenesis of 1) first two arginines (340, 341) to alanine and leucine and 2) leucine (343) to alanine was carried out. Overexpression of these two different mutant versions of Far10 in the wild type yeast strains did not result in toxicity. Moreover, cycloheximide chase analyses of N(Myc)19Far10(L343A) expressed from the endogenous promoter on the chromosome showed that this mutant protein was not stabilized in wild type yeast strains. These data suggest that this sequence in Far10 may not confirm to a classical D-box and that the degradation signals might be located else where in the protein. It could also be possible that mutations in this D-box have to be collective in order for the desired effect(s) to be seen. Database analysis of FAR10 revealed an N-terminal FHA (fork head associated) domain and a C-terminal transmembrane domain. Cell fractionation experiments as well as immunofluorescence studies proved that Far10 localizes to the nuclear envelope [Velten, 2000]. To investigate the function of the C-terminal transmembrane domain, a deletion construct containing Far10 lacking the transmembrane domain, far10?TM was generated. In contrast to wild type Far10 this mutant protein was unable to cause synthetic dosage effects in pre1-1 pre4-1, cdc23-1 and cdc20-1 mutants. Immunofluorescence studies of Far10?TM(HA)2 revealed that this mutant protein was indeed mislocalized. These results provide evidence that the ability of Far10 to induce lethality depends on its correct localization to the nuclear membrane [Murray, 2001]. An investigation into the synthetic interactions of FAR10 with cdc20-1 mutant revealed that cdc20-1 far10? double mutants displayed a synthetic growth defect at 25°C. On the other hand far10? cdc23-1 double mutants showed no obvious growth effects when compared to cdc23-1 single mutants. The data imply that APC is fully active at 25°C in far10? cdc23-1 mutants. On the contrary cdc20-1 far10? double mutants at the same temperature may show a defective APC activity. These results propose that when APC-Cdc20 activity is disturbed, presence of Far10 is required. The mitotic exit network [MEN] in budding yeast is a complex signaling cascade consisting of Tem1 (a GTPase); Cdc15, Dbf2 and Cdc5 (protein kinases); Cdc14 (a protein phosphatase); Mob1 (a Dbf2 associated factor); Bub2-Bfa1/Byr4 (a two component GTPase-activating Protein; GAP); Lte1 (a guanine nucleotide exchange factor; GEF) and a scaffold protein, Nud1 [Amon, 2001]. Tem1 is a positive regulator of MEN. The ultimate effector of MEN is Cdc14 and it is held inactive in the nucleolus by its inhibitor Cfi1/Net1 during G1, S, G2 and early M phase. MEN is activated when the spindle pole body reaches daughter cell where Lte1 (GEF) exchanges a GDP for GTP on Tem1. Thus activated, Tem1-GTP binds to and activates Cdc15, which in turn activates Mob1-Dbf2 complex. Dbf2 facilitates release of Cdc14 from the nucleolus. The freed Cdc14 functions to shut down mitotic Cdk activity by promoting expression of the Cdk inhibitor Sic1 and stimulation of degradation of the essential mitotic cyclins. To outline the relation(s) of FAR10 with regulatory modules of the mitotic exit network a genetic method was executed. For this purpose, effects of FAR10 overexpression and inactivation were studied in MEN mutants. Overexpression of FAR10 in a string of MEN mutants was found to cause toxicity in cdc14-3, dbf2-2, and tem1-3 mutants, with cdc15-2 and cdc5-1 mutants displaying a mild effect and lte1? mutant showing no effect at all. These results prove that when MEN activation is defective cells become sensitive to Far10 overexpression. In contrast when MEN is hyperactive as in the case of bub2? mutants, the effect of overexpression of FAR10 is suppressed. FAR10 is not an essential gene and its deletion causes no obvious growth defects when compared to wild type (W303) strain. Though far10? cdc14-3 double mutants showed no detectable growth effects at either 25°C or 30°C when compared to cdc14-3 single mutants, deletion of FAR10 in dbf2-2 mutants had a moderate suppression effect at 25°C. In the case of far10? tem1-3 double mutants this suppression effect was enhanced at 32°C revealing that FAR10 may be an inhibitor of mitotic exit. Overexpression of FAR10 causes synthetic dosage effects in mutants that are defective in Clb-CDK inactivation such as hct1-?1 and sic1-?1. These results suggest that a defective Clb-CDK inactivation either to due impaired degradation or absence of inhibition by Sic1 makes cells susceptible to Far10 overexpression. In addition, hct1-?1 far10? and sic1-?1 far10? double mutants showed synthetic growth defect at 30°C, which was markedly enhanced at 37°C. The data prove that presence of Far10 is required under these conditions. The ultimate function of the mitotic exit network in budding yeast is inactivation of the mitotic Clb2-CDK activity, which is followed by cytokinesis resulting in the formation of two daughter cells [Visintin et al., 1998]. In relevance of these findings it was rationalized that an enhancement in Clb-CDK inactivation through ectopic overexpression of Sic1 might alleviate the toxic effects associated with FAR10 overexpression. Henceforth, SIC1(HA)1X was co-overexpressed with FAR10 in cdc23-1, cdc14-3, dbf2-2 and tem1-3 mutants. Results in this case show that co-overexpression of SIC1(HA)1X along with FAR10 did not restore wild type growth rates. An analogous result was obtained when Sic1 was overexpressed in MEN mutants that harbored a deletion of FAR10. The data here propose an ill-defined role for Far10 as an inhibitor of mitotic exit. Additionally, these results also provide evidence that FAR10 may act in parallel to MEN and/or co-operate in triggering exit from mitosis.