Abstract. Originally identified as a basolateral domainspecific integral plasma membrane protein of the rat hepatocyte, CE9 mRNA and protein were also detected at high levels in the testis of the rat by Northern and Western blotting and immunoprecipitation . CE9 proved to be a domain-specific integral plasma membrane protein of the rat spermatozoon : on testicular spermatozoa, it was concentrated within the posterior tail domain of the plasma membrane, whereas on vas deferens spermatozoa, CE9 was concentrated within the anterior tail domain . This change in the localization of CE9

Abstract. CE9 is a posterior-tail domain-specific integral plasma membrane glycoprotein of the rat testicular spermatozoon. During epididymal maturation, CE9 undergoes endoproteolytic processing and then redistributes into the anterior-tail plasma membrane domain of the spermatozoon (Petruszak, J. A. M., C. L. Nehme, and J. R. Bartles. 1991. J. Cell. Biol. 114:917-927). We have determined the sequence of CE9 and found it to be a Type Ia transmembrane protein identical to the MRC OX-47 T-cell activation antigen, a member of the immunoglobulin superfamily predicted to have two immunoglobulin-related loops and three asparagine-linked glycans disposed extracellularly. Although encoded by a single gene and mRNA in the rat, the majority of spermatozoal CE9 is of smaller apparent molecular mass than its hepatocytic counterpart due to the under-utilization of sites for asparagine-linked glycosylation. By fluorescence recovery after photobleaching, CE9 was determined to be mobile within the posterior-tail plasma membrane domain of the living rat testicular spermatozoon, thus implying the existence of a regional barrier to lateral diffusion that is presumed to operate at the level of the annulus. Through the development of an in vitro system, the modification of this diffusion barrier to allow for the subsequent redistribution of CE9 into the anterior-tail domain was found to be a time-, temperature-, and energy-dependent process.C ELLS with asymmetrical shapes compartmentalize their integral plasma membrane proteins to morphologically distinguishable surface domains. The mammalian spermatozoon has at least four compositionally distinct plasma membrane domains, corresponding to those covering the anterior and posterior segments of its head and tail (for example see Koehler, 1978;Friend, 1982;Primakoff and Myles, 1983;Holt, 1984). This highly asymmetrical cell compartmentalizes its plasma membrane proteins not only during spermiogenesis, but also through a series of processing and redistribution events that accompany posttesticular maturation in the epididymis, capacitation, and the acrosome reaction Jones et al., 1990;Phelps et al., 1990;Cowan et al., 1991).We recently identified a domain-specific plasma membrane protein of the rat spermatozoon called CE9 (Petruszak et al., 1991). Compartmentalized initially to the posteriortall plasma membrane domain of the testicular spermatozoon, CE9 redistributes into the anterior-tall plasma membrane domain during maturation in the epididymis. This change in localization is preceded by the endoproteolytic Cheryl L. Nehme and Mario M. Cesario contributed equally to this work.Please address reprint requests to Dr. J. R. Bartles, Department of CMS Biology, Northwestern University Medical School, Ward Building, 303 East Chicago Avenue, Chicago, IL 60611.removal of a portion of the amino terminus of the protein.In an effort to learn more about CE9 and its compartmentalization, we have determined the structure of rat CE9, measured its lateral diffusion within the posterior-tall plasma mem...

We have compared the levels of the integral plasma membrane glycoprotein CE9 (MRC OX-47) in different tissues of the rat and have ascertained that the levels of CE9 protein and mRNA in selected tissues and cells exhibit moderate increases in response to diverse stimuli of metabolic activation. When normalized on the basis of total protein, the level of CE9 detected in the different tissues was found to vary over a 50-fold range. In addition, the apparent molecular mass of CE9 was observed to vary from 40 kDa to 68 kDa as a consequence of tissue-specific glycosylation. The highest level of CE9 was detected in brown adipose tissue, where the protein was found to be localized to the plasma membranes of the adipocytes. The metabolic activation of brown adipose tissue that occurs upon exposure of rats to the cold was found to be accompanied by 3.0 +/- 0.4-fold and 1.7 +/- 0.2-fold increases in the levels of CE9 mRNA and protein respectively. An intermediate level of CE9 was detected in the liver, where the protein is known to be expressed within the basolateral domain of the hepatocyte plasma membrane. The metabolic activation of hepatocytes that occurs upon administration of thyroid hormone to euthyroid rats was found to be accompanied by 2.2 +/- 0.3-fold and 1.9 +/- 0.3-fold increases in the levels of CE9 mRNA and protein respectively. A low level of CE9 was detected in the lymphoid organs, such as thymus and spleen. The metabolic activation of isolated rat splenocytes that occurs upon concanavalin A-mediated blast transformation in culture was found to be accompanied by 2.1 +/- 0.2-fold and 1.6 +/- 0.2-fold increases in the levels of CE9 mRNA and protein respectively. On the basis of these and other observations, we suggest that the level, and possibly also the localization, of the integral plasma membrane glycoprotein CE9 may be correlated in a positive fashion with metabolic activity in a diverse array of cell types.

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