Worm Breeder's Gazette 12(3): 65 (June 15, 1992)

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.

Structure/Function Analysis Reveals the Importance of the Membrane Spanning Domains for mec-4 Activity

Kyon-Soo Hong, Maud Kinnell, Monica Driscoll

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 is normally required for the function of the six touch receptor neurons. Rare dominant alleles of this gene induce the cell autonomous swelling and degeneration of the touch receptors. In order to gain insight into the normal function of mec-4 and into how aberrant forms of this protein act to induce cell death, we have begun a structure/function analysis of mec-4 .

The mec-4 gene encodes a protein of about 746 amino acids that has two possible membrane-spanning domains located close to its amino terminal (MSDI) and carboxy terminal (MSDII) ends. Because mec-4 (d)alleles appear to alter osmotic balance in the touch receptor cells, we have suggested that mec-4 may normally function to regulate membrane permeability, perhaps as a subunit of an ion channel.

In order to determine which elements of the mec-4 primary sequence are essential for normal mec-4 function, we are sequencing 50 EMS-induced recessive mec-4 alleles. Because we are most interested in the nature of the "degeneration domain" (the region next to MSDII that includes Ala-442 ,the amino acid that is altered by all degeneration-inducing alleles), we elected to begin our analysis by sequencing the final three exons of the mec-4 gene. These exons encode the last 129 amino acids of the protein and include 1) a highly conserved region, 2) the degeneration domain, 3) MSDII and 4) the carboxy terminus. Most mutations in this part of the protein fall within the membrane-spanning domain. More importantly, the amino acids defined by point mutations to be required for normal mec-4 function are charged or polar residues within this hydrophobic domain. It is interesting that in a helical wheel plot, the glutamate residues in and just outside of the membrane-spanning domain are predicted to line up on one face of the alpha helix; the polar residues are also predicted to be aligned, and fall on the same side of the alpha helix as the charged residues. What we can conclude so far is that residues within MSDII are important for function. We speculate that the negatively charged and polar residues may line a channel that imports positively charged species. Alternatively, this face of the alpha helix may be necessary for association with another protein in a multisubunit complex. We are using site-directed mutagenesis to further probe the importance of the charged amino acids within MDSII for mec-4 function.

We have also sequenced many alleles in the region of MSDI. Unlike MSDII, MSDI is not an amphipathic helix. We have identified one ts mutation that maps to this membrane-spanning domain and alters its hydrophobic character by substituting a charged amino acid.

We have noticed that there are two potential Ca++ binding regions present in the putative extracellular domain of mec-4 (thanks to J. Yuan and B. Horvitz, who sent us a preprint of their ced-4 paper). Because Ca++ influx has been associated with cell death and because the mec-4 MSDII most resembles other cationic channels, we wonder whether Mec-4 could normally act to allow Ca++ influx.