E1 Ubiquitin-Activating Enzyme UBA-1 Plays Multiple Roles throughout C. elegans Development Madhura Kulkarni1, Harold E. Smith2* 1 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America, 2 Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland, United States of America Abstract Poly-ubiquitination of target proteins typically marks them for destruction via the proteasome and provides an essential mechanism for the dynamic control of protein levels. The E1 ubiquitin-activating enzyme lies at the apex of the ubiquitination cascade, and its activity is necessary for all subsequent steps in the reaction. We have isolated a temperature- sensitive mutation in the Caenorhabditis elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Manipulation of UBA-1 activity at different developmental stages reveals a variety of functions for ubiquitination, including novel roles in sperm fertility, control of body size, and sex-specific development. Levels of ubiquitin conjugates are substantially reduced in the mutant, consistent with reduced E1 activity. The uba-1 mutation causes delays in meiotic progression in the early embryo, a process that is known to be regulated by ubiquitin-mediated proteolysis. The uba-1 mutation also demonstrates synthetic lethal interactions with alleles of the anaphase-promoting complex, an E3 ubiquitin ligase. The uba-1 mutation provides a sensitized genetic background for identifying new in vivo functions for downstream components of the ubiquitin enzyme cascade, and it is one of the first conditional mutations reported for the essential E1 enzyme in a metazoan animal model. Citation: Kulkarni M, Smith HE (2008) E1 Ubiquitin-Activating Enzyme UBA-1 Plays Multiple Roles throughout C. elegans Development. PLoS Genet 4(7): e1000131. doi:10.1371/journal.pgen.1000131 Editor: Mary Mullins, University of Pennsylvania School of Medicine, United States of America Received February 13, 2008; Accepted June 18, 2008; Published July 18, 2008 Copyright: 2008 Kulkarni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by National Science Foundation grant number 0445684 to HES. Competing Interests: The authors have declared that no competing interests exist. * E-mail: smithh@umbi.umd.edu Introduction Post-translational modification of proteins performs a critical role in regulating protein activity, and ubiquitin-mediated proteolysis has emerged as the key player in the control of protein turnover. Ubiquitin, a highly conserved small protein, is covalently attached to a target protein through an enzymatic cascade, and the assembly of a poly-ubiquitin chain typically specifies that protein for rapid degradation via the 26S proteasome [1]. Ubiquitin- mediated proteolysis thus provides an ‘‘off’’ switch for governing the spatial and temporal distribution of proteins that are no longer needed. This mode of regulation is essential for normal cellular processes (e.g., cell cycle progression and differentiation), and defects have been implicated in human diseases such as cancers and neurodegenerative disorders [2,3]. Ubiquitination of target proteins can also regulate function by mechanisms other than proteasome-mediated degradation. Mono- ubiquitination serves a signal for endocytosis and trafficking of various cell surface proteins, and is also implicated in histone and transcription factor regulation [4–6]. The assembly of poly- ubiquitin chains can occur at different lysines within ubiquitin, which promotes different outcomes for the labeled protein. Conjugation at lysine 48 typically leads to proteasomal degrada- tion, while linkage through lysine 63 can modulate protein activities in processes as diverse as nuclear localization, DNA repair, or inclusion formation in neurodegenerative diseases [7–9]. A trio of enzymes mediates the attachment of ubiquitin to substrate protein: the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase [10]. Repeated cycles of ligation to the initial ubiquitin lead to poly- ubiquitination. Substrate specificity is conferred by the selective binding of individual E3 ligases to one or a few target proteins [11]. Eukaryotes typically possess a single gene encoding the E1- activating enzyme, tens of E2-conjugating enzymes, and as many as several hundred E3 ligases. Some E3 ligases are themselves multi-subunit complexes, in which a substrate recognition subunit specifies the protein targeted for ubiquitination. In vivo roles for ubiquitination in organismal development have been determined primarily through the characterization of specific E3 ligases. In the nematode Caenorhabditis elegans, E3 ligases regulate processes as diverse as sex determination, cell cycle progression, and synaptic signaling [12–16]. Studies of E2 conjugating enzymes indicate interactions with multiple E3s, as their relative numbers would predict. For example, inactivation of ubc-2 produces a broader range of phenotypes than inactivation of its known E3 partner apc-11 [17]. One of the best-characterized functions for ubiquitination and proteasomal degradation in C. elegans is the coordination of early events of embryogenesis [18]. The anaphase-promoting complex (APC) is a multi-subunit E3 ligase that is essential for completion of meiosis immediately after fertilization of the oocyte by the sperm [19,20]. Ubiquitin-mediated proteolysis also plays a role in the degradation of several proteins that are involved in establishment of anterior-posterior (A-P) polarity in the early embryo. These proteins become asymmetrically localized at the first cell division, and failure to degrade these components correlates with PLoS Genetics | www.plosgenetics.org 1 July 2008 | Volume 4 | Issue 7 | e1000131