Molecular Complex of Three Testis-Specific Isozymes Associated with the Mouse Sperm Fibrous Sheath: Hexokinase 1, Phosphofructokinase M, and Glutathione S-Transferase mu class 5

Mammalian sperm require ATP for motility, and most of it is generated by glycolysis. The glycolytic enzymes segregate to the principal piece region of the flagellum, where some are bound tightly to a novel cytoskeletal structure defining this region, the fibrous sheath (FS), and others are easily extracted with detergents. One of the latter is the spermatogenic cell-specific variant isozyme of hexokinase type 1 (HK1S), characterized by an N-terminal 24-amino acid spermatogenic cell-specific region (SSR). Yeast two-hybrid screens carried out using the SSR as bait determined that HK1S is tethered to muscle-type phosphofructokinase (PFKM) in the principal piece region. This led to the identification of four testis-specific Pfkm splice variants, one that overlapped a variant reported previously (Pfkm_v1) and three that were novel (Pfkm_v2, Pfkm_v3, and Pfkm_v4). They differ from Pfkm transcripts found in somatic cells by encoding a novel 67-amino acid N-terminal extension, the testis-specific region (TSR), producing a spermatogenic cell-specific PFKM variant isozyme (PFKMS). An antiserum generated to the TSR demonstrated that PFKMS is present in the principal piece and is insoluble in 1% Triton X-100 detergent. In subsequent yeast two-hybrid screens, the TSR was found to interact with glutathione S-transferase mu class 5 (GSTM5), identified previously as a spermatogenic cell-specific component of the FS. These results demonstrated that HK1S is tethered in the principal piece region by PFKMS, which in turn is bound tightly to GSTM5 in the FS.