Abstract
In mammalian spermatozoa, most of the type II alpha isoform of cAMP-dependent protein kinase (PKAII alpha) is anchored at the cytoplasmic surface of a specialized array of mitochondria in the flagellar cytoskeleton. This places the catalytic subunits of PKAII alpha in proximity with potential target substrates in the cytoskeleton. The mechanism by which PKAII alpha is anchored at the outer surface of germ cell mitochondria has not been elucidated. We now report the cloning of a cDNA that encodes a novel, germ cell A kinase anchor protein (AKAP) designated S-AKAP84. S-AKAP84 comprises 593 amino acids and contains a centrally located domain that avidly binds regulatory subunits (RII alpha and RII beta) of PKAII alpha and PKAII beta. The 3.2-kilobase S-AKAP84 mRNA and the cognate S-AKAP84 RII binding protein are expressed principally in the male germ cell lineage. Expression of S-AKAP84 is tightly regulated during development. The protein accumulates as spermatids undergo nuclear condensation and tail elongation. The timing of S-AKAP84 expression is correlated with the de novo accumulation of RII alpha and RII beta subunits and the migration of mitochondria from the cytoplasm (round spermatids) to the cytoskeleton (midpiece in elongating spermatids). Residues 1-30 at the NH2 terminus of S-AKAP84 constitute a putative signal/anchor sequence that may target the protein to the outer mitochondrial membrane. Immunofluorescence analysis demonstrated that S-AKAP84 is co-localized with mitochondria in the flagellum.
Highlights
Multiple isoforms of cAMP-dependent protein kinase (PKA)1 are expressed during mammalian spermatogenesis (1–3)
The bulk of the RII␣ polypeptide is attached to the outer membrane of specialized “germ cell” mitochondria that form a helical array around the outer dense fibers and axoneme in the “midpiece” of the flagellum
The expression pattern is consistent with the appearance of S-AKAP84 mRNA during a late phase of spermatid development. This possibility was addressed by monitoring the expression of S-AKAP84 and its cognate mRNA in purified populations of developing germ cells that correspond to meiotic pachytene spermatocytes, postmeiotic round spermatids, condensing spermatids, and epididymal sperm
Summary
Multiple isoforms of cAMP-dependent protein kinase (PKA)1 are expressed during mammalian spermatogenesis (1–3). Residues 1–30 at the NH2 terminus of S-AKAP84 constitute a putative signal/anchor sequence that may target the protein to the outer mitochondrial membrane. Cloning and Sequence Analysis of cDNAs Encoding a Novel AKAP—A human testis cDNA library in bacteriophage gt11 was screened for the expression of -galactosidase fusion proteins that bind 32P-labeled RII with high affinity.
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