Worm Breeder's Gazette 13(1): 39 (October 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.
The rab family of GTP-binding proteins is thought to regulate vesicle trafficking in the cell. We have been investigating the function of rab3 ,a member of this family that is attached exclusively to synaptic vesicles by a polyisoprenyl membrane anchor. Von Mollard et al. (Nature 349:79) have demonstrated that rab3 disassociates from synaptic vesicles upon their fusion at presynaptic terminals. The protein is most likely involved in maintaining unidirectional targeting of synaptic vesicles to the active zone. Previously, we cloned the gene encoding the C. elegans homolog of rab3 and generated antibodies against the protein. These antibodies detect rab3 in the synaptic rich regions of the nervous system. Furthermore, we previously generated two dominant mutations in rab-3 and demonstrated that transgenic animals expressing the mutant rab-3 were variably affected, exhibiting uncoordination, aldicarb resistance, or L1 arrest and uncoordination (1993 meeting, pg. 240). Based on the aldicarb resistant and Unc phenotypes, we now have isolated recessive chromosomal mutations in the rab-3 gene.
We reasoned that we would be able to isolate hypomorphic (and perhaps null alleles) that were viable, uncoordinated and aldicarb resistant when in trans to a deficiency of the rab-3 locus (such as maDf4 ).We crossed mutagenized wild-type N2 males to dpy-25 ( e817sd )/maDf4animals. The young cross progeny were placed onto aldicarb. All classes of progeny should die except animals carrying either a dominant aldicarb resistant mutation, or a mutation in rab-3 that renders the animal resistant to aldicarb. Surviving F1 non-Dpy cross progeny were cloned. 75 independent lines were established in this manner in a screen of roughly 150,000 F1 cross progeny. Using our antibodies, we screened these lines for rab-3 mutants by determining if the strains still expressed rab-3 protein. Four mutant strains were identified that either failed to stain with rab3 antibodies, or had mis-localized rab3 protein.
We have now genetically and molecularly characterized two of the four strains and shown that they carry mutations in the rab-3 locus. rab-3 ( y250 )and rab-3 ( y251 )both have similar phenotypes. The animals are slightly sluggish, slightly jerky during backwards movement, and partially Egl. The phenotypes become more pronounced, but remain mild at 25° C. Males remain capable of mating. Using the aldicarb resistant and antibody staining phenotypes, we have mapped the two mutations to near bli-2 by two factor mapping, and mapped the mutations left of unc-104 by three factor crosses, a region consistent with the location of rab-3 on the physical map. rab3 antibody staining is reduced dramatically in y250 ,while in y251 the rab-3 protein is mis-localized to cell bodies. Molecularly, both alleles are missense mutations in highly conserved regions of the rab-3 gene. y250 changes S er165 to Leu. The serine residue is located in a highly conserved region of GTP binding proteins involved in interacting with the guanine base. y251 changes G ly80 to Glu. This glycine residue is found in the middle of an 8 amino acid region [WDTAGQER] found in all known rab family members that is thought to be involved in stabilization of the gamma-phosphate of GTP. The phenotype of animals carrying either mutation in trans to a deficiency is qualitatively no more severe than homozygous mutant animals. We therefore suspect that mutants carrying a homozygous rab-3 (null)will be similar in phenotype to these mutants. We are in the process of characterizing the two other alleles we have isolated, and are also isolating additional alleles in a non-complementation screen.
In yeast, mutations in other rab family members ypt-1 and sec-4 lead to a lethal null phenotype suggesting that those rab members are absolutely required for proper vesicle trafficking. The surprisingly mild phenotype of our rab-3 mutants suggests the possibility that the rab3 protein plays a less crucial role in directing synaptic vesicle trafficking perhaps due to the nature of the release signal at synapses. We believe we have ruled out the alternative possibility that a redundant rab-3 gene is present in C. elegans (see accompanying WBG article). However, preliminary experiments suggest that rab-3 does play a significant role in mediating some aspects of the vesicle release process, since y250 is synthetically lethal in combination with certain other presynaptic aldicarb resistant mutations.