Caveolin, an integral membrane protein, is a principal component of caveolae membranes in vivo. Two isoforms of caveolin have been identified: a slower migrating 24-kDa species (alpha-isoform) and a faster migrating 21-kDa species (beta-isoform). Little is known about how these isoforms differ, either structurally or functionally. Here we have begun to study the differences between these two isoforms. Microsequencing of caveolin reveals that both isoforms contain internal caveolin residues 47-77. In a second independent approach, we recombinantly expressed caveolin in a caveolin-negative cell line (FRT cells). Stable transfection of FRT cells with the full-length caveolin cDNA resulted in the expression of both caveolin isoforms, indicating that they can be derived from a single cDNA. Using extracts from caveolin-expressing FRT cells, we fortuitously identified a monoclonal antibody that recognizes only the alpha-isoform of caveolin. Epitope mapping of this monoclonal antibody reveals that it recognizes an epitope within the extreme N terminus of caveolin, specifically residues 1-21. These results suggest that alpha- and beta-isoforms of caveolin differ in their N-terminal protein sequences. To independently evaluate this possibility, we placed an epitope tag at either the extreme N or C terminus of full-length caveolin. Results of these "tagging" experiments clearly demonstrate that (i) both isoforms of caveolin contain a complete C terminus and (ii) that the alpha-isoform contains a complete N terminus while the beta-isoform lacks N-terminal-specific protein sequences. Mutational analysis reveals that these two isoforms apparently derive from the use of two alternate start sites: methionine at position 1 and an internal methionine at position 32. This would explain the approximately 3-kDa difference in their apparent migration in SDS-polyacrylamide electrophoresis gels. In addition, using isoform-specific antibody probes we show that caveolin isoforms may assume a distinct but overlapping subcellular distribution by confocal immunofluorescence microscopy. We discuss the possible implications of these differences between alpha- and beta-caveolin.

During spermiogenesis (the maturation of spermatids into spermatozoa) in many vertebrate species, protamines replace histones to become the primary DNA-packaging protein.It has long been thought that this process is facilitated by the hyperacetylation of histone H4. However, the responsible histone acetyltransferase enzymes are yet to be identified. CDY is a human Y-chromosomal gene family expressed exclusively in the testis and implicated in male infertility. Its mouse homolog Cdyl, which is autosomal, is expressed abundantly in the testis. Proteins encoded by CDY and its homologs bear the ''chromodomain,'' a motif implicated in chromatin binding. Here, we show that (i) human CDY and mouse CDYL proteins exhibit histone acetyltransferase activity in vitro, with a strong preference for histone H4; (ii) expression of human CDY and mouse Cdyl genes during spermatogenesis correlates with the occurrence of H4 hyperacetylation; and (iii) CDY and CDYL proteins are localized to the nuclei of maturing spermatids where H4 hyperacetylation takes place. Taken together, these data link human CDY and mouse CDYL to the histone-to-protamine transition in mammalian spermiogenesis. This link offers a plausible mechanism to account for spermatogenic failure in patients bearing deletions of the CDY genes.histone acetylation ͉ spermiogenesis ͉ chromodomain ͉ infertility T he acetylation of N-terminal lysine residues in core histones has been implicated in three distinct cellular processes. The first is the deposition of free histones onto newly synthesized DNA (1). The second is the regulation of gene expression (reviewed in ref.2). The third is the displacement of histones by transition proteins and protamines during vertebrate spermatogenesis (3-6). Various histone acetyltransferase (HAT) enzymes involved in the first two processes have been characterized. HATs involved in the third process have yet to be identified.In mammals and many other vertebrates, dramatic chromatin remodeling occurs during spermiogenesis, whereby histones are displaced from chromatin, first by transition proteins and later by protamines (7). With protamines, DNA in mature spermatozoa is packaged into an extremely condensed, functionally inert configuration (8). Several lines of evidence led to the view that chromatin remodeling during spermiogenesis is facilitated by hyperacetylation of histone H4. First, studies of numerous vertebrate species have shown that H4 hyperacetylation correlates with the occurrence of the histone-to-protamine transition in spermatogenesis. Extensive H4 hyperacetylation occurs in the testes of species where histones are replaced by protamines during spermiogenesis (3-6), but not in species that completely retain somatic-type histones in mature spermatozoa (4, 9). Second, the timing of H4 hyperacetylation is consistent with its role in promoting histone displacement. Studies in the rat, for instance, demonstrated that H4 hyperacetylation during spermiogenesis immediately precedes the histone-to-protamine transition (5, 6). Finally...