SUMO Protease SMT7 Modulates Ribosomal Protein L30 and Regulates Cell-Size Checkpoint Function

Abstract

Proliferating cells actively coordinate growth and cell division to ensure cell-size homeostasis; however, the underlying mechanism through which size is controlled is poorly understood. Defect in a SUMO protease protein, suppressor of mat3 7 (SMT7), has been shown to reduce cell division number and increase cell size of the small-size mutant mating type locus 3-4 (mat3-4), which contains a defective Chlamydomonas retinoblastoma tumor suppressorrelated protein. Here we describe development of an in vitro SUMOylation system using Chlamydomonas components and use it to provide evidence that SMT7 is a bona fide SUMO protease. We further demonstrate that the SUMO protease activity is required for supernumerous mitotic divisions of the mat3-4 cells. In addition, we identified RIBOSOMAL PROTEIN L30 (RPL30) as a prime SMT7 target and demonstrated that its SUMOylation is an important modulator of cell division in mat3-4 cells. Loss of SMT7 caused elevated SUMOylated RPL30 levels. Importantly, overexpression of the translational fusion version of RPL30-SUMO4, which mimics elevation of the SUMOylated RPL30 protein in mat3-4, caused a decrease in mitotic division and recapitulated the size-increasing phenotype of the smt7-1 mat3-4 cells. In summary, our study reveals a novel mechanism through which a SUMO protease regulates cell division in the mat3-4 mutant of Chlamydomonas and provides yet another important example of the role that protein SUMOylation can play in regulating key cellular processes, including cell division.

Revised annotation and extended characterizations of components of the Chlamydomonas reinhardtii SUMOylation system

“…Even so, alignment of sequences of seven SUMO protease domains of Chlamydomonas SUMO proteases share little conservation except the catalytic residues (Figure 6a). The SUMO protease activity of SMT7 (Cre16.g692600) has been demonstrated previously (Lin et al., 2020).…”

“…Cells were harvested from cultures growing in TAP liquid medium. Total RNA was isolated as described previously (Fang, Chung, Chen, Lopez‐Paz, & Umen, 2014; Lin et al., 2020). The full‐length cDNAs of Chlamydomonas SCE2 , SCE3.t2 , SUMO1 , SUMO2 , SUMO3.t1 , E2F1 , DP1 , CDKG1 , CYCD2 , and CYCD3 were amplified by reverse transcription‐polymerase chain reaction (RT‐PCR) using the gene‐specific primers (Table ).…”

“…The full‐length cDNAs of Chlamydomonas SCE2 , SCE3.t2 , SUMO1 , SUMO2 , SUMO3.t1 , E2F1 , DP1 , CDKG1 , CYCD2 , and CYCD3 were amplified by reverse transcription‐polymerase chain reaction (RT‐PCR) using the gene‐specific primers (Table ). The PCR program was carried out as described previously (Lin et al., 2020). The SCE3.t1 cDNA could not be amplified and was omitted from further study.…”

“…Ten nanograms of each pCDFDuet‐SAE1‐SAE2, pETDuet‐SUMO4 GG ‐SCE1, and pRSFDuet‐targets (pRSFDuet‐3XHA‐PCNA, pRSFDuet‐3XHA‐E2F1, pRSFDuet‐3XHA‐DP1, pRSFDuet‐3XHA‐CDKG1, pRSFDuet‐3XHA‐CYCD2, and pRSFDuet‐3XHA‐CYCD3) were co‐transformed into E. coli Rosetta (DE3) by electroporation and selected for the presence of all three plasmids. The in vitro SUMOylation assay was performed as described previously (Lin et al., 2020).…”

“…With availability of haploid genetics, a sequenced genome, mutant libraries, transformation system, gene editing capabilities, and established molecular toolkit (Baek et al., 2016; Blaby et al., 2014; Crozet et al., 2018; Dent, Haglund, Chin, Kobayashi, & Niyogi, 2005; Greiner et al., 2017; Jiang & Weeks, 2017; Kindle, Richards, & Stern, 1991; Kindle, Schnell, Fernandez, & Lefebvre, 1989; Li, Zhang, et al, 2016; Merchant et al., 2007; Picariello et al., 2020; Shin et al., 2016; Tulin & Cross, 2014; Zhang et al., 2014), Chlamydomonas has emerged as a highly valuable single‐cell model system. Chlamydomonas also has a SUMOylation system and SUMO conjugation has been reported to regulate stress responses and the cell division cycle (Knobbe et al., 2015; Lin, Chung, Chen, Chen, & Fang, 2020; Wang et al., 2008). However, an updated comprehensive categorization of the Chlamydomonas SUMO system and its relationship to plant and animal systems remain limited.…”