mec-2: A PUTATIVE REGULATOR OF MEMBRANE PERMEABILITY Mingxia Huang and Marty Chalfie Dept. of Biological Sciences, Columbia University, New York The
mec-2 gene was initially isolated in genetic screens for genes required for touch cell function. The wild-type activity of
mec-2 is also needed for
mec-10-caused touch cell degeneration, suggesting that it may regulate the putative mechanosensory channel complex formed by the gene products of
mec-10,
mec-4, and
mec-6'. We cloned the
mec-2 gene by transposon-tagging and germline rescue of the mutant phenotype. The rescuing activity was first identified in cosmid W05G12 and further minimized to a 17 kb genomic fragment. Several cDNAs were found from mixed- stage libraries constructed by Chris Martin and by Pete Okkema. The longest cDNA (1.8 kb) seems to contain a full- length open reading frame of 440 amino acids that are overall very hydrophilic and highly charged except for a 28 amino acids hydrophobic domain near the N-terminus. The
mec-2 gene appears to be alternatively spliced and some of the pre-mRNAs are not efficiently processed (RT-PCR produced some cDNAs that contained introns). The
mec-2 cDNAs hybridize to several bands on Northern blots of N2 polyA+ RNAs at high stringency (a broad band at 1.8 kb and a weak band between 3 and 4 kb). The larger mRNA is more intense in Northerns of
mec-8(
e398). Thus, a
mec-2 transcript may be a substrate of the putative RNA-binding factor
mec-8 (E. Lundquist and R. Herman, per. comm.). A search of Genbank revealed that
mec-2 is similar to the human gene stomatin (67% identity over the entire 288 amino acid sequence of stomatin). Stomatin is an integral membrane protein that is missing in patients with a rare, autosomal dominant hemolytic anaemia (hereditary stomatocytosis). The red cells in this disease are over hydrated and mouth-shaped and have abnormally high Na+, low K' content. These characteristics have prompted the suggestion that stomatin regulates transmembrane cation influxes2. The stomatin protein is also associated with the red cell cytoskeleton. By analogy the
mec-2 product may have a similar function in the worm touch cells. A
mec-2/acZfusion construct (containing the 77 amino acids N- terminal to the hydrophobic domain
mec-2) is expressed in the six touch cells as well as some other neurons in the head and at the tail. One interesting feature of this staining is that touch cell processes as well as cell bodies stain. The 77 amino acids of
mec-2 within the fusion product have a positive net charge and a predicted helical structure. These are features of some actin- and microtubule-binding proteins. One possible explanation for the process staining is that the N-terminal region of MEC-2 binds to components of the cytoskeleton or its associated proteins in the axons. We are now testing this hypothesis by examining the staining pattern of the
mec-2/acZconstruct in
mec-7and mec-12 mutants, which have defects in the touch cell-specific 15-protofilament microtubules. 1. M. Huang and M. Chalfie (1994) Nature 367, 467-470. 2. G. W. Stewart et al. (1993) Biochim. Biophys. Acta 1225, 15-25.