Worm Breeder's Gazette 12(5): 58 (February 1, 1993)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Update on the Structure/Function Analysis of mec-4

Kyonsoo Hong, Monica Dnscoll

Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, N.J. 08855 (908) 463-5193

mec-4 encodes a protein that is required for the function of the six touch receptor neurons. Rare dominant alleles of this gene induce swelling and degeneration of the touch receptors. To learn more about the normal neuronal funcdon of Mec-4 and the mechanism whereby aberrant forms of cis protein kill cells, we have been conducting a structure/function analysis of mec-4 .

Our characterization of a truncated mec-4 cDNA clone and the comparison of genomic sequences from the mec-4 genes of C. elegans and C briggsae led us to propose that the Mec-4 protein contains two membrane-spanning domains. Because the deduced protein sequence does not include a signal sequence at the amino terminus, we suggested that Mec-4 might be situated such that its amino and carboxyl termini are inside the cell while the rest of the protein faces the extracellular environment.

Mutations in the second membrane-spanning domain block degeneration in cis.

Our analysis of EMS-induced recessive mec-4 mutations has highlighted the functional importance of charged and polar amino acid residues within the second theoretical membrane-spanning domain (WBG 12(3)65). These charged and polar amino acids are predicted to align on one face of an a helix. Such an arrangement of amino acids is characteristic of certain membrane-spanning domains demonstrated to be involved in conductance of known cationic channels. We speculate that Mec-4 may function as a subunit of an ion channel (a mechanosensory channel?) and that the charged and polar amino acids may line the lumen of the channel to facilitate channel conductance. It is interesting that the charged and polar amino acids in the second hydrophobic domain are conserved among various degenerins.

mec-4 alleles bearing large side chain amino acid substitutions for Ala(442) cause the degeneration of the touch receptor neurons. As we are interested in the mechanism of mec-4 (d)-induceddegeneration, we wondered whether single amino acid substations in the membrane-spanning domain could block the death-inducing effects of the large side chain substitutions at position 442. Using site-directed mutagenesis, we introduced single amino acid substitutions in the membrane spanning domain into a mec-4 (d)allele that encodes Val at position 442. These double mutant alleles were introduced into wild type and mec-4 (r)animals to assay their ability to induce degeneration. We have looked at both early and late stage animals and have found no evidence that these alleles induce neuronal degeneration.

According to our channel hypothesis, these results would be consistent with the idea that channel conductance is a necessary aspect of neuronal degeneration. However, it is entirely possible that individual charged and polar amino acids are required for maintaining the structure of the protein or for mediating interaction/assembly of a multisubunit complex. In this scenario, degeneration would not occur because the protein or protein complex is structurally disrupted and is therefore nonfunctional. We are thinking about experimental ways to distinguish between these two models for disruption of the function of Mec-4 by single amino acid substitutions in the membrane-spanning domain.

Another hot-spot for mutations in Mec-4

Our sequence analysis of mec-4 (r)alleles has highlighted an additional region of the protein where amino acid substitutions that disrupt Mec-4 function are clustered. This region is situated in the amino terminal domain that is postulated to be intracellular. It is interesting that this region includes a consensus cAMP-dependent phosphorylation site (two amino acid substitutions disrupt this site, another is a few amino acids away) leading us to wonder whether phosphorylation of Mec-4 could play a role in modulating its cellular activity.