Supplementary Material for: Ccdc33, a Predominantly Testis-Expressed Gene, Encodes a Putative Peroxisomal Protein

Many genes crucial for male fertility are often predominantly or exclusively expressed in male germ cells. The analysis of mouse models has demonstrated the functional importance of peroxisomes in spermatogenesis. The CCDC33 protein has been reported to be a cancer/testis (CT) antigen. We found that mouse Ccdc33 is predominantly expressed in the testis and undergoes alternative splicing to produce at least 4 different transcripts. The protein encoded by Ccdc33 contains 3 coiled-coil domains, a C2-domain, 2 ER membrane retention signal-like motifs and 2 putative peroxisomal targeting signals type 2 (PTS2). We could demonstrate that the second PTS2 sequence is functional and responsible for the targeting of CCDC33 to peroxisomes. Moreover, in HeLa cells CCDC33-dsRED fusion protein co-localized with a known peroxisomal protein, namely PXT1, and showed punctuate intracellular distribution. Taken together, the mouse Ccdc33 encodes a putative peroxisomal protein and is predominantly expressed in male germ cells. The expression starts at the primary spermatocyte stage, suggesting an important role of this protein during spermatogenesis.