SUMOylation Is Developmentally Regulated and Required for Cell Pairing during Conjugation in Tetrahymena thermophila

Abstract

The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena. The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena, including a dynamic regulation associated with the sexual life cycle. P rotein posttranslational modifications are critical regulatory events in eukaryotic cells, adding another layer of complexity to protein function. Modification by ubiquitin (Ub) is known to regulate numerous proteins, most commonly by tagging them for subsequent degradation (reviewed in reference 1). SUMOylation involves the covalent attachment of a small protein called SUMO (small ubiquitin-like modifier) to lysine residues on target proteins. SUMO shares ϳ18% sequence similarity with ubiquitin, and both proteins are relatively small, with similar tertiary structures (reviewed in reference 2). In addition, SUMOylation employs a similar 3-step enzymatic pathway in its conjugation onto target proteins (3-5). SUMO protein, also called Smt3p (suppressor of Mif two 3) in Saccharomyces cerevisiae (6), is first activated by the E1-activating enzyme, a heterodimer consisting of Aos1p and Uba2p, which binds Smt3p via a high-energy thioester linkage in an ATP-dependent step. Activated Smt3p is then transferred to the E2-conjugating enzyme Ubc9p. Several E3 ligases then interact with Ubc9p and direct Smt3p conjugation onto substrates. SUMO-specific proteases (Ulps/SENPs) cleave Smt3p from substrate proteins, making SUMOylati...

Depletion of UBC9 Causes Nuclear Defects during the Vegetative and Sexual Life Cycles in Tetrahymena thermophila

“…More recent studies of Tetrahymena demonstrated that a large increase in SUMOylation occurs during the sexual life cycle when SUMO protein and Uba2p accumulate in the developing macronucleus (24). Although we antic-ipated that depletion of SUMO or Uba2p would result in arrest during macronuclear development, these cells failed to pair, and therefore, later stages of development could not be evaluated (24).…”

“…5). It should be noted that the conditional mutant grown in the absence of CdCl 2 also showed growth, most likely due to leaky expression from the MTT1 promoter, as we have seen in previous studies (24). In other species, Ubc9p has been shown to regulate cell cycle progression, including mitosis and chromosome segregation (14,15,38).…”

“…10), so a large increase upon exposure to CdCl 2 is particularly significant. In addition to Ubc9p effects on pairing, other experiments showed a pairing defect for Uba2p-and Smt3p (SUMO)-depleted cell lines (24). When ⌬UBC9 conditional cells cultured in the absence of CdCl 2 (UBC9 depleted) were mixed with wild-type cells, pairs formed and subsequently entered meiosis (see Fig.…”

“…We recently showed that a large increase in the amount of SUMOylated protein is observed during Tetrahymena sexual reproduction and that the increased signal is centered in the developing macronucleus (24). If this SUMOylation occurs within or around the developing MAC, then the signal for Ubc9p should also accumulate in the anlagen.…”

“…Previous studies demonstrated that RNA interference (RNAi) gene silencing of UBA2 and SUMO in another ciliate, Paramecium tetraurelia, had little effect on vegetative cells but prevented the programmed excision of short regions of DNA called internal eliminated sequences (IESs) during formation of the somatic macronucleus (23). More recent studies of Tetrahymena demonstrated that a large increase in SUMOylation occurs during the sexual life cycle when SUMO protein and Uba2p accumulate in the developing macronucleus (24). Although we antic-ipated that depletion of SUMO or Uba2p would result in arrest during macronuclear development, these cells failed to pair, and therefore, later stages of development could not be evaluated (24).…”