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Model Organisms: New Insights Into Ion Channel and Transporter Function. Stomatin homologues interact in Caenorhabditis elegans

M. M. Sedensky, J. M. Siefker, and P. G. Morgan

Departments of Anesthesiology and Genetics, University Hospitals and Case Western Reserve University, Cleveland, Ohio 44106


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In C. elegans the protein UNC-1 is a major determinant of anesthetic sensitivity and is a close homologue of the mammalian protein stomatin. In humans stomatin is missing from erythrocyte membranes in the hemolytic disease overhydrated hereditary stomatocytosis, despite an apparently normal stomatin gene. Overhydrated hereditary stomatocytosis is characterized by alteration of the normal transmembrane gradients of sodium and potassium. Stomatin has been shown to interact genetically with sodium channels. It is also postulated that stomatin is important in the organization of lipid rafts. We demonstrate here that antibodies against UNC-1 stain the major nerve tracts of Caenorhabditis elegans, with very intense staining of the nerve ring. We also found that a gene encoding a stomatin-like protein, UNC-24, affects anesthetic sensitivity and is genetically epistatic to unc-1. In the absence of UNC-24, the staining of the nerve ring by anti-UNC-1 is abolished, despite normal transcriptional levels of the unc-1 mRNA. Western blots indicate that UNC-24 probably affects the stability of the UNC-1 protein. UNC-24 may therefore be necessary for the correct placement of UNC-1 in the cell membrane and organization of lipid rafts.

lipid rafts; anesthetics; nematodes; development; genetics


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PROTEIN UNC-1 has an important role in determining volatile anesthetic sensitivity in Caenorhabditis elegans (22). UNC-1 is a close homologue of the mammalian protein stomatin, a 31-kDa membrane-associated protein originally described as a component of red cell membranes in humans (32, 33). Several homologues of mammalian stomatin have been identified in the nematode, C. elegans (4, 14, 22). Of these, the protein UNC-1 is the most homologous to mammalian stomatin (22). Mutations in UNC-1 have been shown to affect anesthetic sensitivity in C. elegans as well as cause an uncoordinated phenotype (5, 20). Expression studies using green fluorescent protein (GFP)-UNC-1 fusion and reporter constructs indicate that the unc-1 gene is expressed primarily in the nervous system and throughout postembryonic development (23). The temporal expression pattern is in agreement with earlier studies with temperature-sensitive alleles of unc-1 (12).

In humans, stomatin is missing from erythrocyte membranes in the hemolytic disease overhydrated hereditary stomatocytosis (33). Because the normal transmembrane gradients of sodium and potassium were altered in these erythrocytes, it was first postulated that stomatin played a role in controlling ion flux (32). However, no mutations in stomatin have been associated with families carrying stomatocytosis (7). In addition, mice in which the stomatin gene has been knocked out do not show evidence of any hemolytic phenotype, and their transmembrane ion gradients are not abnormal (38). Thus the precise function of mammalian stomatin is unknown, as is the etiology of stomatocytosis.

Genetic studies with different unc-1 alleles have shown complex intra-allelic interactions, compatible with the protein functioning as an oligomer (21). Consistent with these findings, mammalian stomatin has been shown to exist as a homotypic oligomer composed of 9-12 stomatin molecules (31). Interactions with other gene products have also been shown in both C. elegans and mammals. In C. elegans, UNC-1 interacts genetically with a subunit of the degenerin-type sodium channel, UNC-8 (23). In mammals, stomatin has been found to interact with a G protein-coupled receptor and to colocalize with glycophosphoinositol (GPI)-anchored proteins and lipid rafts (18, 30, 34). Lipid rafts are microdomains in the cell membrane that have increased amounts of sphingolipids and cholesterol. These domains are thought to serve the function of localizing multiple membrane proteins into complexes to ensure proper relative positioning of proteins that physically interact (13). It is known that GPI-anchored proteins and some acylated proteins are localized to these domains. The association of stomatin with GPI-anchored proteins has led to the suggestion that stomatin functions in the formation of lipid rafts and in the trafficking of proteins to the plasma membrane, as described by Snyers et al. (30). This suggestion is supported by the similar structures of stomatin and the lipid-associated protein caveolin. Caveolin is known to be crucial to the formation of caveolae, structures quite similar to lipid rafts (6, 36).

During the study of UNC-1, we found that the distribution of UNC-1 is dependent on a stomatin-like protein (SLP) known as UNC-24. The unc-24 gene was originally characterized by Barnes et al. (4). The UNC-24 protein consists of a stomatin-like domain and a lipid transfer domain. A close homologue of unc-24 has been identified in a cDNA library from human cerebral cortex (28) and has been postulated to be the "brain-specific stomatin" in humans. We find that, in C. elegans, unc-24 controls the distribution or stability of the UNC-1 protein. While lipid rafts have not been described in C. elegans, these studies raise the possibility that unc-24 is responsible for the original establishment of UNC-1 oligomers or lipid rafts. Because both unc-1 and unc-24 affect sensitivity to the lipid-soluble volatile anesthetics, it is possible that these anesthetics interact with lipid rafts.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nematodes. Nematodes were anesthetized as previously described (20), and EC50 values with standard errors were calculated as described by Waud (35). Nematodes were cultured as previously described (23), and all experiments were performed at 20-22°C. The wild-type nematode is N2 in all experiments and was obtained from the Caenorhabditis Genetics Center. The unc-1(X) alleles (e580 and e114), the unc-24(IV) alleles (e138 and eDf28), and the mec-2(X) allele (e1084) were obtained from the Caenorhabditis Genetics Center. The dominant unc-1 alleles n494 and n774 were obtained from Carl Johnson (San Francisco, CA). The unc-1 deletion allele fc53 was isolated in our laboratory (22).

Park and Horvitz (21) originally described the four classes of unc-1. Members of class I (e.g., n494) are semidominant coilers, members of class II (e.g., n774) are semidominant coilers and recessive kinkers, class III alleles (e.g., e114) are hypomorphic recessive kinkers, and class IV alleles (e.g., e580 and fc53) are null-recessive kinkers (the kinkers do not coil, and they move forward more poorly than backward). unc-24 alleles have a kinked motion, similar to that of class III and IV unc-1 alleles (23). The phenotype of the unc-24 mutants is distinguishable from those of the class I and II unc-1 alleles. Thus epistasis between unc-24 and unc-1 could be established.

Genetics. All genetic crosses were performed as described by Brenner (5). The presence of both genes in all double homozygotes was confirmed by noncomplementation tests or, in the case of the dominant alleles, by reisolation of the original homozygous parents from crosses with N2.

UNC-1 protein. The cDNA for UNC-1 was isolated as described by Rajaram et al. (22). The cDNA was subcloned into the multiple cloning site of the pET32A+ vector using the restriction enzymes BamHI and HindIII (Invitrogen). This vector is one of the series of expression vectors containing six tandem histidine residues in the NH2-terminal peptide that have a high affinity for ProBond resin. The his tag was used to purify the expressed protein using the Xpress System from Invitrogen.

Antibodies. Antibodies to UNC-1 were prepared in the laboratory of Man Sun Sy. Two mice were inoculated three times with UNC-1 protein, described above. Once a response was documented from the serum of the mice, hybridomas were made and screened for anti-UNC-1. Seven monoclonal antibodies reacting with UNC-1 were isolated. Each antibody was screened for the ability to bind UNC-1 on a Western blot and for immunocytochemical staining in N2, as well as loss of staining in the unc-1 null alleles fc53 and e580. Five monoclonal antibodies specific for UNC-1 were isolated, each of which gave identical binding in nematodes and on Western blots. One monoclonal antibody, 5D5, was used for all the studies described here. It was used directly from hybridoma growth media. Western blots were done by standard methods (9, 24). Affinity-purified rabbit polyclonal antibody to UNC-17 was a generous gift of Janet Duerr.

Immunocytochemical staining. Immunocytochemical staining of worms was done as described by Duerr et al. (9). All pictured adults are hermaphrodites. Nematodes were prepared with a variation of the freeze-crack method of Albertson (2). Primary antibody incubations (of undiluted monoclonal or 1:50-1:100 polyclonal antibody) were done overnight at 4°C. After being thoroughly rinsed with antibody buffer, slides were incubated in secondary antibody for 4 h at 20°C. Indocarbocyanine (Cy3)-labeled secondary antibodies (donkey anti-mouse and donkey anti-rabbit) were obtained from Jackson ImmunoResearch (West Grove, PA); Oregon Green 488-labeled secondary antibody (goat anti-mouse) was purchased from Molecular Probes. After being rinsed, slides were mounted in antibleaching medium (10) mixed with 4',6-diamidino-2-phenylindole (DAPI) as described by Albertson (1). At least 100 nematodes were examined from each staining experiment, and those shown in Figs. 1-5 are representative examples.


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Fig. 1.   a: staining of N2 (wild type) Caenorhabditis elegans with anti-UNC-1 antibody. Note the pattern of staining primarily in cells of the nervous system. Staining is visible along axons, most notably in the nerve ring (NR), retrovesicular ganglion (RVG), and ventral nerve cord (VNC). P, anterior bulb of the pharynx. b: staining of the anterior portion of a wild-type C. elegans with anti-UNC-1. Note the staining of the NR and VNC (arrows) as noted in a. Also note the pronounced punctate staining of nerve tracts anterior to the NR. c: region of the vulva (V) in a young adult stained with anti-UNC-1. Note the staining of the VNC and the 4 vulva muscles (VM). d: staining of an N2 late embryo with anti-UNC-1. Note the staining of the NR and VNC at this stage of development. e: staining of unc-1(fc53), a null allele of unc-1, with anti-UNC-1 antibody. Note only background staining; no staining of the NR is visible. In addition, no punctate staining is visible along any nerve tracts. Micrographs a, b, d, and e were taken with Zeiss confocal microscope, maximum Z-series projections. Micrograph c was taken with Zeiss Axiophot and Axiocam on a single focal plane. Scale bar, 10 µm.



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Fig. 2.   a: costaining of the VNC with 4',6-diamidino-2-phenylindole (DAPI) and anti-UNC-1. Note the lack of staining in the nuclei (N) and the extension of UNC-1 staining along the VNC. DC, dorsal cord; Sp, sperm. b: costaining of the VNC with anti-UNC-17 (red) and anti-UNC-1 (green). Note that both antibodies similarly stain the nerve tract but that their staining occupies adjacent regions. UNC-1 staining is always more diffuse than that of UNC-17 (vesicular acetylcholine transporter, VAChT). Micrographs were taken with Zeiss Axiophot and Axiocam on a single focal plane. Scale bar, 10 µm.



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Fig. 3.   a: staining of the head of an eDf28 animal with anti-UNC-1 antibody and DAPI. The neuropil of the NR excludes the cell bodies of the ganglia of the head, which are visible as concentrations of blue staining on either side of the space of the NR. This space is ordinarily intensely stained by anti-UNC-1 in N2 (cf. Fig. 1b.). The area of the VNC that lies between cell bodies is not stained, and there is no staining of the DC. We interpret this to indicate that UNC-1 is present but not distributed to the NR. The tail of an adjacent eDf28 hermaphrodite is also seen, with staining of UNC-1 at the pre-anal ganglion (PAG) and lumbar ganglia (LG). b: region of the vulva (V) in a young adult unc-24(eDf28) hermaphrodite stained with anti-UNC-1 (cf. Fig. 1c). Note the diffuse staining of the 4 VM, with no staining surrounding the vulval opening. c: staining of an unc-24 late embryo with anti-UNC-1 antibody. Note the staining of the NR and VNC in a pattern similar to that seen in N2. Micrographs a and c were taken with Zeiss confocal microscope, maximum Z-series projections. Micrograph b was taken with Zeiss Axiophot and Axiocam on a single focal plane. Scale bar, 10 µm.



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Fig. 4.   Costaining of the mid-VNC of unc-24 animals with anti-UNC-1 and DAPI. Note that the level of staining is greatly decreased compared with that seen in N2. In particular, no staining is visible in the VNC away from the nuclei (N, blue). However, punctate dots of staining are noted on each end of many nerve cell nuclei. In general, we found 2 such dots per nucleus. Micrograph was taken with Zeiss Axiophot and Axiocam on a single focal plane. Scale bar, 10 µm.



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Fig. 5.   A: Northern blot of N2, two unc-1 alleles (e580 and fc53), and two unc-24 alleles (e138 and eDf28). All lanes were loaded with 50 ug of total RNA and normalized with an actin probe (not shown). Note that the unc-1 message is transcribed in both unc-24 alleles; thus any effect of unc-24 on unc-1 is not at the level of transcription. The unc-1 message is 1.2-kb in size, consistent with the predicted unc-1 message size and previous Northern blots of poly(A)+ RNA probed with the unc-1 cDNA. B: Western blot of N2, two unc-1 alleles (e580 and fc53), and two unc-24 alleles (e138 and eDf28). Fourteen micrograms of protein were loaded in each lane (as determined by Bio-Rad assay). The UNC-24 protein is not identified on this blot. The predicted size for the UNC-1 protein is 32 kDa and correlates to the upper band (small arrow), whereas the major band in N2 is at 24 kDa (large arrow). The other minor bands may represent cross-staining of other stomatin-like proteins with anti-UNC-1. Note that the amount of UNC-1 (large arrow) is decreased or absent in both the unc-1 and unc-24 alleles. Coupled with the data in Figs. 3 and 4, this finding indicates that the UNC-1 protein either is not translated efficiently or is unstable in the absence of UNC-24.

Northern blots. Mixed stage RNA was prepared from C. elegans by standard methods (25) using RNAzol. These RNAs were prepared from N2, two unc-1 alleles and two unc-24 alleles. Fifty micrograms of total RNA were loaded in each lane, electrophoresed on formaldehyde gels, and transferred to nylon membrane (3). The Northern blots were probed with the full-length unc-1 cDNA and washed in 50% formamide at 42°C. Because the cultures were from mixed-stage cultures, equal loading of RNA in each lane was assured by probing with the cDNA from the actin gene act-1 (gift of M. Krause, Washington DC) (16).

Western blots. Total protein samples were prepared by boiling 1- to 2-g pellets of nematodes in SDS with protease inhibitor, followed by sonication as described by Duerr et al. (9). Samples were centrifuged, and the supernatant was isolated. Protein samples were run on 10% SDS-polyacrylamide gels. The concentrations of protein samples were determined using the Bio-Rad protein assay kit. The resulting concentrations were used to ensure that 14 ug of the resulting protein were loaded in each lane. All Western blot results were repeated in duplicate using separate protein preparations and were done on separate days.

Microscopy. Pictures of antibody-stained animals were taken with a Zeiss Axioplan with confocal microscope (model 600; Bio-Rad). Pictures of a single focal plane of antibody-stained animals were taken with a Zeiss Axiophot microscope equipped for fluorescence, using a Zeiss Axiocam digital camera.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Staining wild-type C. elegans with anti-UNC-1 antibody showed a punctate distribution of protein along nerve tracts. Staining was most intense in the nerve ring, the retrovesicular ganglion, and the ventral and dorsal nerve cords (Fig. 1, a and b). Staining was also prominent in the nerve tracts of the snout (Fig. 1b), around the vulva (Fig. 1c), and along the sublateral nerve tracts of the body. No staining was seen in other tissues. Staining was noted in late embryos in the nerve ring and ventral nerve cord (Fig. 1d). Incubation of UNC-1 antibody with fc53, a null allele of unc-1, showed no staining (Fig. 1e). The staining of N2 was consistent with the fluorescence reported previously in live unc-1(0) animals rescued with a GFP-UNC-1 fusion construct (23). Antibody staining of the dominant unc-1 allele n494 showed a pattern similar to that for N2; however, the class II allele n774 showed less overall staining than N2 in a grossly normal distribution (data not shown). Staining with anti-UNC-1 was tested in animals mutant in each of three other genes that alter anesthetic sensitivity: unc-79, unc-8, and gas-1. In addition, two other mutants, unc-7 and unc-9, which cause a kinked phenotype like unc-1, were stained with anti-UNC-1. Staining was grossly normal in each of these mutant animals (data not shown).

The cellular distribution of UNC-1 was localized by costaining with DAPI, for nuclear staining, and by costaining with anti-UNC-17 (vesicular acetylcholine transporter, VAChT), a marker of cholinergic synapses. Costaining by anti-UNC-1 and DAPI showed that UNC-1 was not found in the nucleus but, rather, was distributed along the presumed axons of neurons (Fig. 2a). This staining extended to the dorsal nerve cord and sublateral nerve tracts. Costaining with anti-UNC-17 showed that both UNC-1 and UNC-17 occupied adjacent regions in the ventral and dorsal nerve cords; however, they do not colocalize (Fig. 2b). UNC-1 was expressed in a more diffuse pattern in the ventral and dorsal nerve cords than UNC-17.

Mutations that cause visible phenotypes in two other genes encoding stomatin-like proteins, mec-2 and unc-24, have been identified. We stained mec-2(0) and unc-24(0) animals to demonstrate that the antibody did not cross-react with these proteins. Staining of mec-2(e1084) animals was not significantly different from that of N2 (data not shown). However, in unc-24 (both eDf28 and e138) animals, staining with anti-UNC-1 was dramatically changed compared with that in N2 (Fig. 3a). Sparse punctate staining could be seen in unc-24 animals primarily over the ganglia of the head, with basically no staining in the area of the nerve ring. This staining was much fainter than the staining of the nerve ring in N2 and was never seen in unc-1(0) animals. The distribution of another neuron-specific protein, UNC-17, was not changed from that of N2 in unc-24 animals (data not shown). Thus this change in UNC-1 antibody staining was not the result of a general disruption of the structure of the nervous system. Staining of the vulval muscles was present, but there was no staining that delineated the vulval opening (Fig. 3b, compare with Fig. 1c). Staining in unc-24 embryos was similar to that of N2 embryos of the same age (Fig. 3c). Costaining of unc-24 animals with anti-UNC-1 and DAPI revealed a striking pattern. UNC-1 staining appeared to consist of two punctate dots per cell at opposite ends of the nucleus (Fig. 4) and is especially easy to see in the ventral nerve cord. No staining was noted along the axons or in the dorsal nerve cord, which contains no cell bodies.

Northern blots of unc-1(0) and unc-24(0) animals, probed with an unc-1 cDNA, show that the unc-1 gene was transcribed into a 1.2-kb message in N2 and unc-24 animals but not in unc-1(0) animals (Fig. 5A). Probing with the actin gene act-1 showed equal loading in each lane (data not shown). We then probed Western blots of the same strains with the anti-UNC-1 antibody. These blots showed decreased staining of the predominant protein in both unc-1 and unc-24 animals compared with N2 (Fig. 5B). The predominant protein identified by anti-UNC-1 is smaller than the predicted UNC-1 protein (24 kDa observed compared with 32 kDa predicted and seen in the UNC-1 expression construct). A fainter band is seen at the predicted size in N2 and is also present in e580, e138, and eDf28.

Because there was an apparent physical interaction between the UNC-24 protein and the UNC-1 protein, we determined whether there was also a genetic interaction. Loss-of-function alleles of both unc-24 and unc-1 share similar phenotypes, i.e., kinked uncoordinated motion, suppression of unc-79, and increased sensitivity to the volatile anesthetic diethylether (Table 1). However, the dominant allele unc-1(n494) exhibits a coiled motion, quite different from the kinked motion seen with unc-1 loss-of-function alleles. In addition, n494 has an increased sensitivity to several volatile anesthetics, including halothane (23). The molecular defect identified in n494 is a missense mutation changing a glycine to an arginine at position 182 (G182R), which leads to an altered function for the protein but does not eliminate protein expression. We constructed the double mutant unc-24(eDf28);unc-1(n494) to test for epistasis. We found that unc-24 was fully epistatic to n494 in that the double mutant had a kinked phenotype and a normal sensitivity to halothane, as does unc-24(eDf28) alone (Table 1). This finding indicates that unc-24 function is necessary for the n494 phenotype. In addition, the double mutant unc-24(eDf28);unc-1(n494) is able to fully suppress the abnormal sensitivity of unc-79 to halothane (data not shown). The double mutant showed a staining for UNC-1 similar to that for unc-24 alone, while n494 alone stains normally compared with N2 (data not shown). A second dominant mutant, unc-1(n774), exhibits a coiled phenotype as a heterozygote and a kinked phenotype as a homozygote. unc-24(eDf28) is also epistatic to n774/+ [i.e., unc-24(eDf28);unc-1(n774)/+ is kinked]. The effects of eDf28 on the anesthetic sensitivity of n774 were not determined. Finally, we tested the halothane sensitivity of the double mutant containing both the unc-24 null allele eDf28 (normal halothane sensitivity) and the unc-1 null e580 (slight halothane resistance). The double mutant had normal halothane sensitivity (data not shown). In all cases described, the double mutants adopted the phenotype resulting from the unc-24 mutation alone. Thus unc-24 is epistatic to unc-1.

                              
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Table 1.   Effects of unc-24(eDf28) on sensitivity to volatile anesthetics


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibody staining indicates that UNC-1 is widely expressed in the nervous system of C. elegans. This staining is in a punctate pattern along the major nerve tracts of the animal. The amino acid sequence of UNC-1 indicates that it is membrane bound. A close UNC-1 homologue, mammalian stomatin, has been localized to the plasma membrane (31, 32). Both anti-UNC-1 and anti-UNC-17 intensely stain the nerve ring of C. elegans. However, in the ventral and dorsal nerve cords, the two proteins appear to lie near each other but do not overlap. UNC-1 staining always appears more diffuse, especially over the ventral nerve cord. Therefore, we do not think that UNC-1 is localized to a synapse. UNC-1 is most likely present in the plasma membrane of axons in C. elegans.

The loss of staining for UNC-1 in unc-24 animals indicates that UNC-24 probably affects the expression or stability of the UNC-1 protein. The Northern blots show that this effect is not at the level of transcription. The Western blot indicates that either the UNC-1 message is not translated into protein or that the protein is degraded in the absence of UNC-24. The predicted size for the UNC-1 protein is 32 kDa, whereas the major band in N2 is at 24 kDa. The fact that the dominant band in N2 is at a lower size probably indicates that the UNC-1 protein undergoes posttranslational modification by removal of some portion of the protein. However, we cannot rule out the possibility that the actual translated sequence is shorter than the predicted sequence. The minor band at 32 kDa may indicate the presence of the complete protein. Interestingly, the 32-kDa band is still present in the genetic null allele e580. The phenotype seen in e580 may result from the improper processing of the UNC-1 protein.

In unc-24 mutants, the localization of UNC-1 protein close to cell nuclei is most consistent with the hypothesis that UNC-24 affects the trafficking of UNC-1 to the cell periphery. Because the endoplasmic reticulum lies adjacent to the cell nucleus, it is possible that UNC-24 is necessary for movement of UNC-1 from the endoplasmic reticulum to the cell membrane. Snyers et al. (29, 30) showed that stomatin lies within juxtanuclear vesicles and the plasma membrane in mammalian cells in tissue culture. They also demonstrated that stomatin localizes to lipid rafts in these cells. Lipid rafts are microdomains in the cell membrane containing groups of proteins, sphingolipids, and cholesterol (13, 30). The punctate distribution of UNC-1 is certainly consistent with its placement in discrete regions rather than uniformly distributed in the cell membrane. Snyers et al. postulates that stomatin normally cycles from the perinuclear region to the plasma membrane in association with other specialized membrane proteins. It may be that UNC-24 controls movement of UNC-1 to proper membrane localization in nerve cells. Whether this function is specific for UNC-1 is unknown, although we have shown that unc-24(0) does not change the pattern of UNC-17 staining as it does for UNC-1. We have preliminary data showing that all stomatin recognized by the monoclonal antibody used in this study localizes to lipid rafts in C. elegans (Morgan and Sedensky, unpublished observations) in wild-type animals. Whether unc-24 mutations alter the normal pattern of UNC-1 localization to lipid rafts is unknown. However, given the apparent inability of UNC-1 to travel away from the nucleus in an unc-24(0) background, we would predict that the distribution of UNC-1 will be different from lipid rafts in unc-24(0) compared with N2 animals. The failure of UNC-1 to be transported to the periphery could, in turn, either affect its stability or, via a feedback mechanism, stop further synthesis of the protein. Because stomatin is known to form homotypic oligomers (31), and since UNC-24 is a stomatin-like protein (4), it is also possible that UNC-24 is a necessary component for the initial formation or for the maintenance of such multimeric interactions. Such protein interactions may, in turn, affect the stability of UNC-1. The absence of UNC-1 protein on Western blots from unc-1 animals indicates that UNC-24 is not merely responsible for exposure of the UNC-1 epitope to the antibody. Rather, the UNC-1 protein is largely absent in unc-24 animals.

The suggestion that unc-24 is epistatic to unc-1 is consistent with the above findings. Because UNC-1 is not distributed in a functional pattern in the absence of UNC-24, the dominant alleles of unc-1 (n494 and n774) cannot express UNC-1 in a manner able to cause the dominant phenotype. (We have stained eDf28;n494 and e138;n494 with anti-UNC-1 antibody and found that these double mutants stain like the unc-24 alleles alone.) This epistasis is not allele specific, because both alleles of unc-24 are epistatic to at least three different alleles of unc-1, one each from classes I, II, and IV (21). Class III alleles of unc-1 are not able to be tested for epistasis because their phenotypes are essentially the same as those of null alleles of unc-24. The double mutant unc-24(eDf28);unc-1(n494) probably results in an unc-1 loss of function phenotype, because, like an unc-1(0), the double mutation suppresses the abnormal sensitivity of unc-79 to halothane (data not shown). However, as noted in RESULTS, we did not detect the slight halothane resistance seen in the class IV null unc-1 allele e580.

It is possible that heterooligomers of stomatin are responsible for stomatin function and staining in the wild-type animal. Ten different genes coding for SLPs have been identified by the genome sequencing project (37). However, it is not clear that all the genes are expressed, and mutations causing altered phenotypes have only been identified in three of these genes. Nevertheless, it remains possible that these other proteins may affect the function of UNC-1. If different heterooligomers of SLPs exist, they may have varied effects in subsets of cells. Additionally, antibody staining of UNC-1 may depend on the specific interactions between UNC-1 and multiple other proteins including SLPs. Other SLPs that interact with UNC-1 may be identified in screens for suppressors and enhancers of unc-1.

In C. elegans, UNC-1 and SLPs also have an effect on sensitivity to volatile anesthetics (20, 22, 23). We have previously shown that unc-1 genetically interacts with unc-8 and another gene, unc-79, to alter anesthetic sensitivity (23). It is important to note that while these studies did find a genetic interaction, no evidence indicates a direct interaction of the protein products. As with unc-1(0), unc-24 is a suppressor of the abnormal halothane sensitivity of unc-79 (Table 1). Because UNC-24 affects the correct distribution of UNC-1, it probably affects the protein products of these other genes indirectly through UNC-1.

Obviously, it will be of interest to know the temporal expression pattern of UNC-24 to help determine how UNC-24 affects UNC-1. It is interesting to recall that stomatin is absent from the membranes of erythrocytes from patients with stomatocytosis, despite having an apparently normal stomatin gene (7, 32, 33). It is possible that defects in another protein product, similar to the effects seen with UNC-24 in nematodes, are responsible for the absence of stomatin from erythrocytes. A close homologue of UNC-24 has been isolated from a human brain cDNA library (28), but its function remains unknown.

The potential localization of UNC-1 to lipid rafts is also interesting when anesthetic sensitivity is considered. A long-known feature of volatile anesthetics is that their potencies correlate with their lipid solubilities. This led to a long-held hypothesis that membrane lipids were the targets of volatile anesthetics. Yet, most theories now postulate that volatile anesthetics directly interact with protein. Lipid rafts are intriguing candidates to reconcile two sets of data regarding anesthetic sites of action. Because volatile anesthetics are lipid soluble, they could partition into lipid rafts and affect the lipid-protein complexes that are present. It is presently not known whether volatile anesthetics partition differently into such rafts than into the remaining cell membrane. However, small but significant differences in potency exist between stereoisomers of volatile anesthetics (8, 26, 27). These differences have been interpreted to indicate that volatile anesthetics have their primary effects on proteins (8, 11). Because sphingolipids and cholesterol have chiral centers and localize to lipid rafts (13, 30), the differences between different stereoisomers of some volatile anesthetics may relate to interactions not only with proteins but also with the lipid and cholesterol composition of the surrounding milieu.

It is interesting that several membrane components postulated to affect anesthetic sensitivity (stomatins, G protein-coupled receptors, syntaxin-related proteins, nitric oxide synthase, and others) have all been noted to be associated with lipid rafts (15, 17, 19, 30). Such rafts may represent a unifying physical property that allows anesthetics access to such membrane-associated protein complexes. These lipid-protein complexes are potentially a widespread group of similar targets for volatile anesthetics.


    ACKNOWLEDGEMENTS

We are indebted to Janet Duerr for assistance with antibody staining techniques and generous sharing of the anti-UNC-17 antibody. We also thank Man Sun Sy for direction and assistance in making monoclonal antibodies. We appreciate discussions with Gordon Stewart with regard to the work on stomatin. In addition, we thank Ernst-Bernhard Kayser, Shanta Rajaram, Helen Salz, and Peter Harte for support and ongoing discussions.


    FOOTNOTES

These studies were supported by National Institute of General Medical Sciences Grants GM-45402 and GM-58881.

Address for reprint requests and other correspondence: P. G. Morgan, Dept. of Anesthesiology, 2400 Bolwell Bldg., Univ. Hospitals, 11100 Euclid Ave., Cleveland, OH 44106 (E-mail: pgm2{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 May 2000; accepted in final form 6 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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