MOLECULAR CHARACTERIZATION OF A SMALL HEAT-SHOCK ALPHA-CRYSTALLIN PROTEIN IN ENCYSTED ARTEMIA EMBRYOS

Molecular chaperones protect cells during stress by limiting the denat uration/aggregation of proteins and facilitating their renaturation. I n this context, brine shrimp embryos can endure a wide variety of stre ssful conditions, including temperature extremes, prolonged anoxia, an d desiccation, thus encountering shortages of both energy (ATP) and wa ter. How the embryos survive these stresses is the subject of continui ng study, a situation true for other organisms facing similar physiolo gical challenges. To approach this question we cloned and sequenced a cDNA for p26, a molecular chaperone specific to oviparous Artemia embr yos. p26 is the first representative of the small heat shock/alpha-cry stallin family from crustaceans to be sequenced, and it possesses the conserved alpha-crystallin domain characteristic of these proteins. Th e secondary structure of this domain was predicted to consist predomin antly of beta-pleated sheet, and it appeared to lack regions of alpha- helix. Unique properties of the nonconserved amino terminus, which sho wed weak similarity to nucleolins and fibrillarins, are enrichments in both glycine and arginine. The carboxyl-terminal tail is the longest yet reported for a small heat shock/alpha-crystallin protein, and it i s hydrophilic, a common attribute of this region. Site-specific differ ences between amino acids from p26 and other small heat shock/alpha-cr ystallin proteins bring into question the functions proposed for some of these residues. Probing of Southern blots disclosed a multi-gene fa mily for p26, whereas two size classes of p26 mRNA at 0.7 add 1.9 kilo base pairs were seen on Northern blots; the larger probably representi ng nonprocessed transcripts, Examination of immunofluorescently staine d samples with the confocal microscope revealed that a limited portion of intracellular p26 is found in the nuclei of encysted embryos and t hat it resides within discrete compartments of this organelle. The res ults in this paper demonstrate clearly that p26 is a novel member of t he small heat shock/alpha-crystallin family of proteins. These data, i n concert with its restriction to embryos undergoing oviparous develop ment, suggest that p26 functions as a molecular chaperone during expos ure to stress, perhaps able; to limit protein degradation and thus ens ure a ready supply of functional proteins when growth is reinitiated.