The tomato MADS box gene no. 5 (TM5) is shown here to be expressed in meristematic domains fated to form the three inner whorls-petals, stamens, and gynoecia-of the tomato flower. TM5 is also expressed during organogenesis and in the respective mature organs of these three whorls. This is unlike the major organ identity genes of the MADS box family from Antirrhinum and Arabidopsis, which function in overlapping primordial territories consisting of only two floral whorls each. The developmental relevance of the unique expression pattern of this putative homeotic gene was examined in transgenic plants. In agreement with the expression patterns, antisense RNA of the TM5 gene conferred both early and late alterations of morphogenetic markers. Early defects consist of additional whorls or of a wrong number of organs per whorl. Late, organ-specific changes include evergreen, cauline, and unabscised petals; green, dialytic, and sterile anthers; and sterile carpels and defective styles on which glandular trichomes characteristic of sepals and petals are ectopically formed. However, a complete homeotic transformation of either organ was not observed. The early and late floral phenotypes of TM5 antisense plants suggest that TM5 mediates two unrelated secondary regulatory systems. One system is the early function of the floral meristem identity genes, and the other system is the function of the major floral organ identity genes.

RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.A growing number of RING (really interesting new gene) finger proteins have been implicated as key regulators of cell growth and in organ and tissue development (1). RINGs are cysteine-rich, zinc-binding domains defined by a pattern of conserved cysteine and histidine residues (reviewed in reference 1) that exert their regulatory function through association with and effects on signal transduction and cell cycle control proteins. In many cases RINGs elicit E3 ligase activity, which serves to limit their availability and to alter the stability and/or localization of their associated proteins (1,4,18,20).RINGs that directly target ubiquitination of their associated substrates via their E3 ligase activity include the mammalian homologues of seven in absentia (Siah), AO7, BRCA1, and Mdm2 (1,12,18,20); Parkin, which is associated with autosomal juvenile parkinsonism (36); and the Cbl protein family, which regulates cell signaling (18, 37). In the process of searching for RING finger proteins resembling mammalian RINGs known to exhibit E3 ligase activities in Caenorhabditis elegans, we identified and characterized RNF5 (C16C10.7), the C. elegans homologue of the human RING-finger protein 5 (RNF5) (17) and Arabidopsis thaliana RMA1 (17, 22). RNF5 shows structural homology within the RING domain with BRCA1, Cbl, and Mdm2 and is important to development in C. elegans (Broday et al., unpublished data). RNF5 expression is reduced in human tumors (our unpublished observations), partly due to transcriptional suppression mediated by the polycomb group protein EZH2, which is involved in prostate cancer progression (44). The search for RNF5-associated proteins identified a LIM domain-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that through its association ...

The small ubiquitin-like modifier (SUMO) modification alters the subcellular distribution and function of its substrates. Here we show the major role of SUMO during the development of the Caenorhabditis elegans reproductive system. smo-1 deletion mutants develop into sterile adults with abnormal somatic gonad, germ line, and vulva. SMO-1ϻGFP reporter is highly expressed in the somatic reproductive system. smo-1 animals lack a vulval-uterine connection as a result of impaired ventral uterine -cell differentiation and anchor cell fusion. Mutations in the LIN-11 LIM domain transcription factor lead to a uterine phenotype that resembles the smo-1 phenotype. LIN-11 is sumoylated, and its sumoylation is required for its activity during uterine morphogenesis. Expression of a SUMO-modified LIN-11 in the smo-1 background partially rescued -cell differentiation and retained LIN-11 in nuclear bodies. Thus, our results identify the reproductive system as the major SUMO target during postembryonic development and highlight LIN-11 as a physiological substrate whose sumoylation is associated with the formation of a functional vulval-uterine connection.[Keywords: SUMO; somatic gonad; Supplemental material is available at http://www.genesdev.org. (Schwarz et al. 1998). SUMO conjugation has been shown to affect subcellular localization of the modified substrate, thereby affecting its activity and stability (Matunis et al. 1996;Mahajan et al. 1997; Muller at al. 1998). Several transcription factors are modified by sumoylation. Whereas SUMO modification negatively regulates the androgen receptor, SP3, c-Jun, and p53 (Gostissa et al. 1999; Muller et al. 2000;Poukka et al. 2000;Schmidt and Muller 2002), sumoylation of the glucocorticoid receptor increases its transcriptional activities (LeDrean et al. 2002). Sumoylation also affects transcriptional activities indirectly. For example, SUMO conjugation to class II histone deacetylase impairs its transcription-repressing function (Kirsch et al. 2002). Alternatively, sumoylation has also been shown to affect nuclear and subnuclear (nucleolar or PML nuclear body) localization of regulatory proteins primarily implicated in transcriptional control (Sternsdorf et al. 1997;Pichler et al. 2002).The SUMO conjugation system is essential for viability in Saccharomyces cerevisiae (Melchoir 2000). Phenotypes observed upon aberrant sumoylation in S. cerevisiae include impaired septin ring formation, chromosomal segregation, and progression of the cell cycle through G 2 -M (Johnson and Blobel 1999). Studies in Arabidopsis suggest that the SUMO conjugation system has a role in protection against stress and/or repair of stressrelated damage (Kurepa et al. 2002). In Drosophila melanogaster, the loss-of-function mutation of semushi, the UBC9 (SUMO-conjugating enzyme) ortholog, prevents nuclear import of the transcription factor Bicoid (Bcd) and results in impaired embryogenesis (Epps and Tanda 1998).

Summary Sumoylation is a reversible post-translational modification that plays roles in many processes, including transcriptional regulation, cell division, chromosome integrity and DNA damage response. Using a proteomics approach, we identified ~250 candidate targets of sumoylation in C. elegans. One such target is the cytoplasmic intermediate filament (cIF) protein named IFB-1, which is expressed in hemidesmosome-like structures in the worm epidermis and is essential for embryonic elongation and maintenance of muscle attachment to the cuticle. In the absence of SUMO, IFB-1 formed ectopic filaments and protein aggregates in the lateral epidermis. Moreover, depletion of SUMO or mutation of the SUMO acceptor site on IFB-1 resulted in a reduction of its cytoplasmic soluble pool, leading to a decrease in its exchange rate within epidermal attachment structures. These observations indicate that SUMO regulates cIF assembly by maintaining a cytoplasmic pool of non-polymerized IFB-1, and that this is necessary for normal IFB-1 function.

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with—and regulation of its ubiquitination and stability by—RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5−/− MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5−/− using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5−/− mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5−/− macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.

Activating transcription factor 2 (ATF2/CRE-BP1) is implicated in transcriptional control150-248 in fibroblasts or melanoma but not in ATF2-null cells caused a profound G 2 M arrest and increased degree of apoptosis following irradiation. The interaction between ATF2 and TIP49b constitutes a novel mechanism that serves to limit ATF2 transcriptional activities and highlights the central role of ATF2 in the control of the cell cycle and apoptosis in response to stress and DNA damage.

Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/β-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle–muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.

Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin-proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies.HMG-CoA synthase | sterol synthesis | yeast two-hybrid