Primary structure of a novel 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS)-binding membrane protein highly expressed in Torpedo californica electroplax.
Polyclonal rabbit antibodies were raised against 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), an inhibitor of a variety of anion transport proteins. These antibodies specifically recognize SITS-reacted erythrocyte band 3 in immunoprecipitations and Western blots. In Western blots of SITS-reacted membrane proteins derived from vesicles of the electric organ of Torpedo californica (known to express a SITS-sensitive Cl- channel) the antibodies recognized two major species of approximately 93 kDa and approximately 105 kDa. The approximately 93 kDa protein was identified as the alpha-subunit of the Na,K-ATPase. The approximately 105 kDa protein (designated sp105) is a glycoprotein which binds to wheat-germ agglutinin and concanavalin A and is present as a disulphide-linked homodimer under non-reducing conditions. A partial amino acid sequence and a polyclonal antibody were used to clone the corresponding cDNA. sp105 is encoded in electroplax by two abundant mRNAs of approximately 6 and approximately 6.8 kb. A hybridizing mRNA of approximately 5 kb was over 200-fold and over 500-fold less abundant in brain and heart respectively. Sequence analysis of the cDNA predicted a novel protein of 697 amino acids containing eight potential N-linked glycosylation sites. Analysis of hydrophobicity indicated the presence of at least one, and possibly three, putative membrane-spanning domains. When expressed from the Sp6 message in Xenopus laevis oocytes, the protein was inserted into membranes, glycosylated and processed to form a dimer. However, no increase in 36Cl uptake or in membrane conductance could be detected. We found no effect of hybrid depleting the specific message on expression of the Torpedo electroplax Cl- channel in oocytes. Thus we conclude that this novel electroplax membrane protein is probably not a functional part of the chloride channel.
Whitehead Institute for Biomedical Research, 9 Cambridge Center, MA 02142.
T J
TJ
Jentsch
A M
AM
Garcia
H F
HF
Lodish
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000302:isolation & purification*; D004557:Electric Organ / Q000032:analysis*; D006023:Glycoproteins; D008565:Membrane Proteins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D014101:Torpedo / Q000378:metabolism*
D002352:Carrier Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; C060764:SITS protein, Torpedo californica