Characterization of T4 Mutants That Partially Suppress the Inability of T4rII to Grow in Lambda Lysogens

In the course of isolating viable T4 deletions that affect plaque morphology (Homyk and Weil 1974), two closely linked point mutants, sip1 and sip2, were obtained. They map between genes t and 52, cause a reduction in plaque size and burst size, and partially suppress the lethality of rII mutants for growth in lambda lysogens. These characteristics demonstrate that sip1 and sip2 are similar to mutants previously reported by Freedman and Brenner (1972). In addition, D. Hall (personal communication) has shown that sip1 and sip2 are similar to the mutant farP85, which affects the regulation of a number of early genes ( Chace and Hall 1975).——Sip suppression of rII mutants can be demonstrated in one-step growth experiments, even when both rII genes are completely deleted. This indicates that sip mutants do not simply reduce the level of rII gene products required for growth in a lambda lysogen. Instead, they alter the growth cycle so as to partially circumvent the need for any rII products.——Mutations at two other sites, designated L₁ and L₂, reverse the poor phage growth caused by sip and, in the one case tested, reverse the rII-suppressing ability of sip.     

Using RNA Interference to Identify Specific Modifiers of a Temperature-Sensitive,Embryonic-Lethal Mutation in the Caenorhabditis elegans Ubiquitin-Like Nedd8 Protein Modification Pathway E1-Activating Gene rfl-1

The essential Caenorhabditis elegans gene rfl-1 encodes one subunit of a heterodimeric E1-activating enzyme in the Nedd8 ubiquitin-like protein conjugation pathway. This pathway modifies the Cullin scaffolds of E3 ubiquitin ligases with a single Nedd8 moiety to promote ligase function. To identify genes that influence neddylation, we used a synthetic screen to identify genes that, when depleted with RNAi, enhance or suppress the embryonic lethality caused by or198ts, a temperature-sensitive (ts) mutation in rfl-1. We identified reproducible suppressor and enhancer genes and employed a systematic specificity analysis for each modifier using four unrelated ts embryonic lethal mutants. Results of this analysis highlight the importance of specificity controls in identifying genetic interactions relevant to a particular biological process because 8/14 enhancers and 7/21 suppressors modified lethality in other mutants. Depletion of the strongest specific suppressors rescued the early embryonic cell division defects in rfl-1(or198ts) mutants. RNAi knockdown of some specific suppressors partially restored Cullin neddylation in rfl-1(or198ts) mutants, consistent with their gene products normally opposing neddylation, and GFP fusions to several suppressors were detected in the cytoplasm or the nucleus, similar in pattern to Nedd8 conjugation pathway components in early embryonic cells. In contrast, depletion of the two strongest specific enhancers did not affect the early embryonic cell division defects observed in rfl-1(or198ts) mutants, suggesting that they may act at later times in other essential processes. Many of the specific modifiers are conserved in other organisms, and most are nonessential. Thus, when controlled properly for specificity, modifier screens using conditionally lethal C. elegans mutants can identify roles for nonessential but conserved genes in essential processes.UBIQUITIN-mediated proteolysis regulates many biological processes (Nandi et al. 2006). In the early Caenorhabditis elegans embryo, these include oocyte maturation, cell cycle progression, cell polarization, and cell fate patterning, all of which require the timely destruction of maternally expressed proteins (Bowerman and Kurz 2006; Greenstein and Lee 2006). One C. elegans protein targeted for proteolysis early in embryogenesis is MEI-1, the AAA-ATPase subunit of the microtubule-severing complex called katanin (Mains et al. 1990; Dow and Mains 1998; Srayko et al. 2000; Kurz et al. 2002; Pintard et al. 2003a; Xu et al. 2003). Katanin is a heterodimer of two subunits called p60 and p80 in vertebrates and MEI-1 and MEI-2 in C. elegans. Katanin in C. elegans is required for proper assembly and function of the small, barrel-shaped meiotic spindles (Albertson and Thomson 1993; McNally et al. 2006) and must be degraded after meiotic divisions to permit assembly of the much larger first mitotic spindle in the one-cell zygote. In mutants that fail to degrade katanin after the completion of meiosis, the first mitotic spindle is fragmented and mis-oriented, cytokinesis is defective, and the embryos die without hatching (Dow and Mains 1998; Srayko et al. 2000; Kurz et al. 2002).The katanin subunit MEI-1 is targeted for poly-ubiquitylation and proteolytic destruction by a Cullin-based E3 ligase (Kurz et al. 2002). This complex includes the Cullin scaffolding protein CUL-3 and a substrate-specific adaptor called MEL-26 that binds to CUL-3 through a BTB domain and to MEI-1 through a MATH domain (Pintard et al. 2003b). Cullin 3-based E3 ligases in mammals also utilize substrate-specific adaptor proteins that, like MEL-26, have both a Cullin-binding BTB/POZ domain and another protein–protein interaction domain that binds to the substrate (Geyer et al. 2003; Cullinan et al. 2004; Angers et al. 2006). While MEI-1/Katanin downregulation by the CUL-3/MEL-26 E3 ligase is essential at most growth temperatures, a mel-26 null mutation is viable at the low growth temperature of 15° (Lu and Mains 2007). This bypass of mel-26 at 15° depends at least in part on the anaphase-promoting complex and its targeting of MEI-1 for proteolytic degradation (Lu and Mains 2007). Phosphorylation by the kinase MBK-2 primes MEI-1 for proteolysis (Quintin et al. 2003; Stitzel et al. 2007) and also promotes the downregulation of MEI-1 by the anaphase-promoting complex (Lu and Mains 2007).CUL-3 is the only C. elegans Cullin thus far identified that requires modification by the ubiquitin-like protein Nedd8 (Bowerman and Kurz 2006). In contrast, C. elegans CUL-2 is required for progression through meiosis and for the localized degradation of cell fate determinants in one-cell-stage embryos (Liu et al. 2004; Sonneville and Gonczy 2004), but neddylation-defective mutants do not exhibit these early defects (Bowerman and Kurz 2006). Cullin neddylation is mediated by the Nedd8 protein conjugation pathway, which begins with a heterodimeric E1-activating enzyme consisting of ULA-1 and RFL-1 (Uba3p in budding yeast) and also includes the E2-conjugating enzyme UBC-12 (Jones and Candido 2000; Srayko et al. 2000; Kurz et al. 2002) and the E3 ligase DCN-1 (Kurz et al. 2005).The downregulation of MEI-1/katanin by the CUL-3/MEL-26 E3 ligase requires a balance of both CUL-3 neddylation, which is mediated by the Nedd8 conjugation pathway, and deneddylation, which is mediated by the conserved COP-9 Signalosome (Pintard et al. 2003a). Other Cullin-based E3 ubiquitin ligases also require a balance of neddylation and deneddylation (Lyapina et al. 2001; Schwechheimer et al. 2001; Bornstein et al. 2006; Hetfeld et al. 2008). Deneddylation may modulate activation of the E3 ligase and thereby prevent the premature degradation of substrate adaptor proteins that also can become poly-ubiquitylated and degraded as a result of E3 ligase function.To identify additional factors that influence neddylation, and the downregulation of MEI-1/katanin after the completion of meiosis in C. elegans, we report here our use of RNA interference (RNAi) to reduce gene functions in a temperature-sensitive (ts) neddylation-defective mutant, rfl-1(or198ts). The discovery of RNAi and its systemic properties in C. elegans have made it possible to systematically target C. elegans genes for depletion by feeding worms bacterial strains that express double-strand RNAs corresponding to C. elegans gene sequences (Fire et al. 1998; Timmons et al. 2001; Feinberg and Hunter 2003; Baugh et al. 2005; Lehner et al. 2006; van Haaften et al. 2006). Furthermore, chemical mutagenesis screens have identified temperature-sensitive mutations in many essential C. elegans genes, which can be used for synthetic screens by choosing intermediate-growth temperatures that sensitize the genetic background and also optimize visual scoring of embryonic viability. Recently, genomewide RNAi screens have been used to identify C. elegans genes that, when reduced in function, restore viability to temperature-sensitive, embryonic-lethal mutants (Labbe et al. 2006; O''Rourke et al. 2007). Because a loss of suppressor function restores mutant viability, the suppressors may negatively regulate either the wild-type gene product or the process that requires the wild-type gene product.Here we report our identification of C. elegans genes that, when reduced in function by feeding RNAi, reproducibly suppressed or enhanced rfl-1(or198ts) embryonic lethality. Most suppressors were specific for rfl-1(or198ts), while specific enhancement was less common. Many of the rfl-1-specific suppressors and enhancers are conserved but appear nonessential. GFP fusions to several specific suppressors exhibit localization patterns that resemble those known for neddylation pathway components, and depletion of some of these partially restored CUL-3 neddylation in rfl-1(or198ts) mutants. In addition to identifying possible roles for conserved genes in cullin neddylation, we report the first quantitative analysis of specificity for both the enhancement and the suppression of a conditionally lethal mutant in C. elegans. Our results highlight the importance of testing genetic modifiers of conditionally lethal mutants for locus specificity.     

Suppressors of Mutations in the rII Gene of Bacteriophage T4 Affect Promoter Utilization

Homyk, Rodriguez and Weil (1976) have described T4 mutants, called sip, that partially suppress the inability of T4rII mutants to grow in λ lysogens. We have found that mutants sip1 and sip2 are resistant to folate analogs and overproduce FH₂ reductase. The results of recombination and complementation studies indicate that sip mutations are in the mot gene. Like other mot mutations (Mattson, Richardson and Goodin 1974; Chace and Hall 1975; Sauerbier, Hercules and Hall 1976), the sip2 mutation affects the expression of many genes and appears to affect promoter utilization. The mot gene function is not required for T4 growth on most hosts, but we have found that it is required for good growth on E. coli CTr5X. Homyk, Rodriguez and Weil (1976) also described L mutations that reverse the effects of sip mutations. L₂ decreases the folate analog resistance and the inability of sip2 to grow on CTr5X. L₂ itself is partially resistant to a folate analog, and appears to reverse the effects of sip2 on gene expression. These results suggest that L₂ affects another regulatory gene related to the mot gene.     

d-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.     

Distal Recognition Sites in Substrates Are Required for Efficient Phosphorylation by the cAMP-Dependent Protein Kinase

Protein kinases are important mediators of signal transduction in eukaryotic cells, and identifying the substrates of these enzymes is essential for a complete understanding of most signaling networks. In this report, novel substrate-binding variants of the cAMP-dependent protein kinase (PKA) were used to identify substrate domains required for efficient phosphorylation in vivo. Most wild-type protein kinases, including PKA, interact only transiently with their substrates. The substrate domains identified were distal to the sites of phosphorylation and were found to interact with a C-terminal region of PKA that was itself removed from the active site. Only a small set of PKA alterations resulted in a stable association with substrates, and the identified residues were clustered together within the hydrophobic core of this enzyme. Interestingly, these residues stretched from the active site of the enzyme to the C-terminal substrate-binding domain identified here. This spatial organization is conserved among the entire eukaryotic protein kinase family, and alteration of these residues in a second, unrelated protein kinase also resulted in a stable association with substrates. In all, this study identified distal sites in PKA substrates that are important for recognition by this enzyme and suggests that the interaction of these domains with PKA might influence specific aspects of substrate binding and/or release.PROTEIN kinases are key mediators of signal transduction in all eukaryotic cells. Each protein kinase modifies a distinct set of substrates, and the biological consequences of activating any kinase are the result of the collective actions of these target proteins (Hunter 2000; Manning et al. 2002). The ability to identify substrates is therefore essential for a complete understanding of most signaling pathways. Unfortunately, this identification process tends to be difficult, and few physiologically relevant targets are known for most protein kinases (Manning and Cantley 2002; Johnson and Hunter 2005). This situation may be changing as a number of innovative approaches to this problem have been developed in recent years (reviewed in Ptacek and Snyder 2006; Deminoff and Herman 2007; Ubersax and Ferrell 2007).This article is focused on the cAMP-dependent protein kinase (PKA) from the budding yeast, Saccharomyces cerevisiae. The PKA enzyme is found in all eukaryotes and is one of the most intensely studied members of this protein family (Taylor et al. 2005). PKA was the first protein kinase structure to be described, and its structure has provided essential insights into the general organization and catalytic mechanism of these enzymes (Knighton et al. 1991; Smith et al. 1999). Subsequent work has illustrated the conserved nature of the protein kinase core and the different ways that the activity of these enzymes can be regulated (Hunter 2000; Huse and Kuriyan 2002; Kannan and Neuwald 2005). In S. cerevisiae, PKA activity is a key regulator of cell growth and the response to environmental stress (Toda et al. 1985; Thevelein and De Winde 1999; Herman 2002; Schneper et al. 2004). We are interested in understanding the role of PKA in these processes and have identified a number of substrates for this enzyme (Howard et al. 2003; Chang et al. 2004; Budovskaya et al. 2005; Deminoff et al. 2006). One of the approaches used for this identification took advantage of PKA variants that exhibit a stable binding to substrate proteins (Deminoff et al. 2006). This binding is novel as most wild-type protein kinases, including PKA, interact only transiently with their substrates (Manning and Cantley 2002). Interestingly, one of these PKA variants was altered at a residue that is conserved in all protein kinases, suggesting that it might be possible to generate substrate-binding versions of other enzymes in this family.These variants of PKA were used here to explore the nature of the protein kinase–substrate interaction. These studies identified substrate domains distal to the sites of phosphorylation that were required for efficient recognition by the wild-type PKA, both in vitro and in vivo. These substrate domains were found to interact with a C-terminal region of PKA that is itself removed from the active site of the enzyme. A systematic mutagenesis of PKA identified additional residues that, when altered, resulted in a stable association with substrates. These latter residues are in close proximity in the three-dimensional structure and may link the active site with this C-terminal substrate-binding domain of PKA. Finally, we show that similar alterations within a second protein kinase, the mammalian double-stranded RNA-dependent protein kinase (PKR), also led to an increased affinity for substrates. In all, the data suggest that the interactions described here may be generally important for protein kinase function and models that explain potential roles for these substrate domains are discussed.