The TPR snap helix: a novel protein repeat motif from mitosis to transcription

The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo

“…We also examined whether the Ski2p-containing complex could be recovered by copurification with tagged Ski3p or tagged Ski8p+ GST-Ski8p expression was induced by the addition of galactose and cells were labeled with [ 35 S]-methionine+ Extracts were prepared and GST-Ski8p was purified by immunoprecipitation with a-GST antibodies+ The proteins in the bound fraction were analyzed by SDS-PAGE and autoradiography+ Under these conditions, a labeled protein of the size expected for GST-Ski8p (70 kDa) was specifically retained (Fig+ 1B)+ In addition, labeled proteins of the sizes expected for Ski2p and Ski3p were also specifically retained when GST-Ski8p was used, but not when wild-type Ski8p without GST was used as a control+ The higher level of radioactivity in the GST-Ski8p band compared to the putative Ski2p and Ski3p bands was due to galactose-induced overexpression of GST-Ski8p+ A similar copurification from labeled cells was done using HA-tagged Ski3p (Ski3p3xHA) expressed from the genomic SKI3 locus+ As predicted, in addition to recovering Ski3p3xHA, a band in the expected position of Ski2p was also observed (Fig+ 1C)+ However, several additional proteins ranging in size from about 40 kDa to 70 kDa were also specifically immunoprecipitated with Ski3p3xHA+ These additional bands were not observed in control experiments using untagged Ski3p (Fig+ 1C) nor were they seen in Western blots of Ski3p3xHA-containing crude extracts (data not shown), suggesting that they were not degradation products of Ski3p+ Because Ski8p migrates in this size range, we could not identify an individual band as Ski8p+ However, we have shown above that Ski8 and Ski3p associate+ The stoichiometry of Ski3p to Ski2p in Figure 1C is approximately 2:1, suggesting that, unlike Ski2p, there is an excess of Ski3p over Ski2p+ This excess Ski3p may then be free to associate with other proteins+ Based on the ideas that TPR and WD-repeat proteins are often found in association with each other (Goebl & Yanagida, 1991;van der Voorn & Ploegh, 1992;Neer et al+, 1994;Smith et al+, 1999) and speculation that Ski3p and Ski8p physically interact (Matsumoto et al+, 1993;Masison et al+, 1995), we examined whether Ski3p and Ski8p could interact in the absence of Ski2p+ Ski3p3xHA was immunoprecipitated from extracts prepared from SKI2 wild-type or ski2⌬ strains and the immunoprecipitates were assayed for GST-FIGURE 1. Coimmunoprecipitation of a protein complex containing Ski2p, Ski8p, or Ski3p+ Yeast strains were labeled with [ 35 S]-methionine+ Extracts were prepared and proteins were immunoprecipitated with tag-specific antibody and protein A beads+ The immunoprecipitated proteins were fractionated by SDS-PAGE on 6-18% acrylamide gradient gels and autoradiographed+ A: Extracts from strains BJ5464 (WT), RKY2033 (ski3⌬ ), RW2911 (ski6-2), and AJY685 (ski8⌬ ) carrying either pAJ39 (SKI2) or pAJ160 (SKI2cmyc) were immunoprecipitated with a-c-myc antibody+ Short arrows identify putative Ski8p+ B: Immunoprecipitations were performed on extracts from strain CH1305 carrying either pAJ267 (SKI8) or pAJ261 (GST-SKI8) using a-GST antibody+ C: Immunoprecipitations were performed on extracts from strains CH1305 (SKI3 ) and AJY245 (SKI3HA ) using a-HA antibody+ The positions of protein bands consistent with being tagged or untagged Ski2p, Ski3p, and Ski8p are indicated+ Additional protein bands in the Ski3p3xHA lane of C that were coprecipitated with Ski3p are noted with asterisks+ ϩ and Ϫ indicate the presence or absence of the respective tag+ Ski8p by Western blott...…”

“…The current models for Ski protein function make certain predictions about their localization+ A role in 39 mRNA degradation would be supported by a cytoplasmic localization and a role in ribosome biogenesis would be supported by a nuclear or nucleolar localization+ In an effort to dissect the general cellular role of the antiviral Ski proteins, we examined the protein interactions of Ski2p, Ski3, and Ski8p and the intracellular localization of Ski2p and Ski3p+ Ski3p is a 164-kDa protein that contains ten copies of a tetratricopeptide repeat (TPR) (Rhee et al+, 1989)+ TPR proteins are typically found in protein complexes and often in association with WDrepeat proteins (Goebl & Yanagida, 1991;van der Voorn & Ploegh, 1992;Neer et al+, 1994;Smith et al+, 1999)+ Ski3p also contains a canonical leucine zipper motif (LX 6 LX 6 LX 6 L) from amino acid residues 1232 to 1253 (J+T+ Brown & A+W+ Johnson, unpubl+ observation), suggesting protein oligomerization+ Ski3p in yeast is reported to be nuclear (Rhee et al+, 1989)+ Ski8p is a 44-kDa protein containing five WD repeats (Matsumoto et al+, 1993;Smith et al+, 1999; "The WD-repeat Family of Proteins" at http://bmerc-www+bu+edu/wdrepeat/)+ Based on the similarity of phenotypes of ski3 and ski8 mutants and the protein families to which these proteins belong, it has been suggested that these proteins physically interact (Matsumoto et al+, 1993;Masison et al+, 1995 Figure 1A were not specific to tagged Ski2p+ Quantitation of the Ski2p and putative Ski3p and Ski8p bands indicated that they were present in a 1:1:1 stoichiometry when accounting for the number of methionines in each species+ We repeated this immunoprecipitation experiment in ski3 and ski8 deletion mutants+ Coimmunoprecipitation of Ski2p in a SKI3 deletion mutant abolished the coprecipitating band migrating in the position expected for Ski3p (Fig+ 1A, ski3⌬ )+ Surprisingly, deletion of SKI3 also abolished the putative Ski8p band+ Similarly, in a SKI8 deletion mutant, the putative Ski3p and Ski8p bands were both absent (Fig+ 1A, ski8⌬ )+ Similar immunoprecipitation experiments performed in ski6-2, ski4-1, and ski7::HIS3 strains showed no effect of these mutations on the Ski2p complex (Fig+ 1A, ski6, and data not shown)+ These results strongly suggest that Ski2p is in a complex with Ski3p and Ski8p and that the interaction of Ski2p with Ski3p and Ski8p requires Ski8p and Ski3p, respectively+ Upon overexposure of the autoradiograph shown in Figure 1A or in additional immunoprecipitation experiments, we did not observe additional proteins coprecipitating specifically with Ski2p+…”

Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition

“…The Drosophila crooked neck (crn) gene resides in region 2E3 of the X chromosome and encodes a 702-amino-acid protein composed almost exclusively of sixteen direct copies of a variant tetratricopeptide repeat (TPR) element (Zhang et al+, 1991)+ Perrimon and colleagues (Zhang et al+, 1991) have shown that the crn gene is expressed throughout the embryo and is present in the larval, pupal, and adult stages+ Null allele mutants of crn die in late embryogenesis with impaired neurological and muscle development (Zhang et al+, 1991;Drysdale et al+, 1993)+ For instance, the horizon-tal commissures of the ventral nerve cord are much thinner than normal and the corresponding longitudinal connectives are reduced or absent+ Likewise, some muscle groups fail to develop and the yolk remains as a fixed plug within the crn mutant embryo+ The ubiquitous gene expression pattern of the crn gene and the embryonic lethal phenotype of null mutants indicate that crn supports a fundamental, essential cellular process+ The gross similarity of the crn TPR elements with similar elements in the fungal cdc16, cdc23, nuc2ϩ, and BimA cell cycle proteins contributed to an early suggestion that crn might be a component of the Drosophila cell cycle machinery (Zhang et al+, 1991)+ However, the crn TPR motif contains distinctive sequence features not found in the cell cycle protein set (Sikorski et al+, 1991)+ It remains uncertain whether crn activity directly contributes to cell cycle progression+ TPR-bearing proteins are present in the three genetic kingdoms and function in support of many cellular processes, such as transcription, peroxisome biogenesis, cell cycle progression and PKR protein kinase inhibition and pre-mRNA splicing (Legrain & Choulika, 1990;Goebl & Yanagida, 1991;Sikorski et al+, 1991;Gindhart & Goldstein, 1996;Urushiyama et al+, 1997;Kyrpides & Woese, 1998)+ Generally, TPR elements are clustered in three or more repeats and act to promote site-specific protein/protein contact+ Although the crn-like TPR motif is not common, the yeast genome codes for several proteins with multiple copies of this element (McLean & Rymond, 1998)+ Among the proteins with the best matches are three RNA processing proteins, two U1 snRNP proteins (Prp39p and Prp42) and a phylogenetically conserved mRNA 39 end processing factor (Rna14p)+ This correlation between the crn-like TPR repeat and RNA processing proteins led us to ask whether the apparent yeast crn counterpart, CLF1, also contributed to RNA metabolism+ Here we present evidence to show that the Clf1p protein is an essential and conserved pre-mRNA splicing factor+ The data support a model in which Clf1p/crn functions as a scaffold to organize the intron and advance spliceosome assembly through its distinctive TPR repeats+ In addition, this study provides evidence that the crn-like TPR motif is restricted to RNA processing proteins and thus a valuable predictor of protein function+…”

The WD‐40 repeat

“…A similar motif has been found in several other proteins. Based on the presence of the WD-40 repeat, these proteins may constitute a WD-40 family.Members of the WD-40 family include: (i) the fl-subunit of guanine nucleotide regulatory proteins (G-proteins), a component of the heterotrimeric complex that transduces signals from transmembrane receptors to a variety of second messenger generating effectors [1,2]; (ii) STE4, a functional G-protein fl-subunit homologue in yeast (Saccharomyces cerevisiae), involved in a signal pathway that controls the response to mating pheromone [3]; (iii) CDC4, a component of the yeast nuclear cytoskeleton, required at the late Gj/S phase boundary of the cell cycle [4,5]; (iv) CDC20, a yeast gene product required for several microtubule-dependent processes at multiple stages in the cell cycle [6][7][8] [24,25]. It should be noted, however, that the second motif in ligninase displays a rather weak homology to the WD-40 consensus sequence.…”

“…Members of the WD-40 family include: (i) the fl-subunit of guanine nucleotide regulatory proteins (G-proteins), a component of the heterotrimeric complex that transduces signals from transmembrane receptors to a variety of second messenger generating effectors [1,2]; (ii) STE4, a functional G-protein fl-subunit homologue in yeast (Saccharomyces cerevisiae), involved in a signal pathway that controls the response to mating pheromone [3]; (iii) CDC4, a component of the yeast nuclear cytoskeleton, required at the late Gj/S phase boundary of the cell cycle [4,5]; (iv) CDC20, a yeast gene product required for several microtubule-dependent processes at multiple stages in the cell cycle [6][7][8] [9]; (vi) I2.3, the product of a gene in the chicken MHC-iocus [10]; (vii) PRP4, a stable component of yeast U4/U6 small nuclear ribonucleoprotein particle (snRNP) [11][12][13]; (viii) PRPI7, another protein involved in pre-mgNA splicing in yeast [13]; (ix) AER~ TUP1, a transcriptional repressor in yeast [14--15]; (x) MSll, a negative regulator of the RAS-cAMP pathway in yeast [16]; (xi) coronin, a component of the actin/ myosin complex of the slimemoid Dict),ostelium discoideum [17]; (xii) PWPI, a yeast protein, containing periodic tryptophan residues [18]; (xiii) Clbp, a Chlano,do. monas protein of unknown function [19]; (xiv) MAKI 1, the apparently membrane associated product of an essential yeast gene, necessary for the maintenance of killer M1 double-stranded RNA [20]; and (xv) AAC3, the deduced product of a developmentally regulated transcript in Dictyosteliunz discoideum that contains long AAC repeats (reading fi'ame specifies glutamine in this case) [21].…”