Short title: Regulation of PP1γ2 by sds22

Testis- and sperm-specific protein phosphatase, PP1γ2, is a key enzyme regulating sperm function. Its activity decreases during sperm maturation in the epididymis. Inhibition of PP1γ2 leads to motility initiation and stimulation. Our laboratory is focused on identifying mechanisms responsible for the decline in PP1γ2 activity during sperm motility initiation in the epididymis. Previously, using immuno-affinity chromatography, we showed that a mammalian homologue of yeast sds22 is bound to PP1γ2 in motile caudal spermatozoa (Huang et al. Biol. Reprod. 2002; 67: 1936). The objectives of this study were to determine: (1) stoichiometry of PP1γ2-sds22 binding and (2) whether PP1γ2 in immotile caput epididymal spermatozoa is bound to sds22. The enzyme from caudal and caput sperm extracts was purified by column chromatography. Immunoreactive PP1γ2 and sds22 from both caudal and caput spermatozoa were found in the flow-through fraction of a DEAE-cellulose column. However, PP1γ2 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active. The DEAE-cellulose flow-through fractions were next passed through a SP-sepharose column. Caudal sperm sds22 and PP1γ2 co-eluted in the gradient fraction. In contrast, caput sperm sds22 an