Current Biology 17, 592–598, April 3, 2007 ª2007 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2007.01.074

ReportLRK-1, a C. elegans PARK8-RelatedKinase, Regulates Axonal-DendriticPolarity of SV Proteins

Aisa Sakaguchi-Nakashima,1 James Y. Meir,3,5

Yishi Jin,3,4 Kunihiro Matsumoto,1,2,*

and Naoki Hisamoto1,2

1Department of Molecular BiologyGraduate School of ScienceInstitute for Advanced ResearchNagoya University2Solution-Oriented Research for Science

and TechnologyJapan Science and Technology CorporationChikusa-kuNagoya 464-8602Japan3Howard Hughes Medical Institute andDepartment of Molecular Cell and Developmental

BiologyUniversity of CaliforniaSanta Cruz, California 950644Howard Hughes Medical Institute andDivision of Biological SciencesSection of NeurobiologyUniversity of California, San Diego9500 Gilman DriveLa Jolla, California 92093

Summary

Neurons are polarized cells that contain distinct setsof proteins in their axons and dendrites. Synaptic

vesicles (SV) and many SV proteins are exclusively lo-calized in the presynaptic regions but not in dendrites.

Despite their fundamental importance, the mecha-nisms underlying the polarized localization of SV pro-

teins remain unclear. The transparent nematode Cae-norhabditis elegans can be used to examine sorting

and transport of SV proteins in vivo. Here, we identifya novel protein kinase LRK-1, a C. elegans homolog of

the familial Parkinsonism gene PARK8/LRRK2 that isrequired for polarized localization of SV proteins. In

lrk-1 deletion mutants, SV proteins are localized toboth presynaptic and dendritic endings in neurons.

This aberrant localization of SV proteins in the den-drites is dependent on the AP-1 m1 clathrin adaptor

UNC-101, which is involved in polarized dendritictransport, but not on UNC-104 kinesin, which is re-

quired for the transport of SV to presynaptic regions.The LRK-1 proteins are localized in the Golgi appara-

tus. These results suggest that the LRK-1 proteinkinase determines polarized sorting of SV proteins

to the axons by excluding SV proteins from thedendrite-specific transport machinery in the Golgi.

*Correspondence: g44177a@nucc.cc.nagoya-u.ac.jp5 Current address: Center for Molecular Chaperone/Radiobiology

and Cancer Virology, Medical College of Georgia, Augusta, Georgia

30912.

Results and Discussion

C. elegans LRK-1 Is Homologous to the Familial

Parkinsonism Gene PARK8/LRRK2Parkinson’s disease (PD) is the most common motorneurodegenerative disorder. The LRRK2/PARK8 genehas been recently identified as a causative gene for au-tosomal dominant PD [1, 2]. The LRRK2 protein belongsto the ROCO family of complex proteins and containsthe following five domains in order: leucine-rich repeats,a Roc (Ras in complex proteins) domain that belongs tothe Ras GTPase superfamily, a COR (C-terminal of Roc)domain, a MAPKKK-like kinase domain, and WD-40repeats (Figure 1A). To address the question of howLRRK functions in a whole organism, we have under-taken a genetic analysis of LRRK function by usingC. elegans as a model system. C. elegans containsonly one lrk-1 gene encoding an LRRK-like protein (Fig-ure 1A). We determined the expression pattern of lrk-1by using a green fluorescent protein (GFP) fusion(lrk-1N::gfp) (Figure 1B) and found that LRK-1N::GFPwas expressed in many tissues, including neurons,hypodermis, muscles, and intestine (Figure 1C). Wegenerated two deletion mutants of the lrk-1 gene(Figures 1A and 1B). Both deletions would result intruncated proteins that were likely to abolish the gene’sfunction. Worms carrying these mutations exhibitedsubtle defects in movement and were partially defectivein chemotaxis to volatile odorants (data not shown).However, as reported below, we also found that LRK-1was required for polarized SV localization.

LRK-1 Regulates Axonal-Dendritic Polarity

of SV Localization

To visualize SV localization in live animals, we useda fluorescently tagged SV marker composed of theC. elegans VAMP2/synaptobrevin protein fused to GFP(SNB-1::GFP) [3] under the control of the flp-13 promoter[4]. In head sensory neurons of adult wild-type animals,the SNB-1::GFP protein was localized strictly to theaxonal regions but not to dendritic processes (Fig-ure 2A). In lrk-1(km17) mutants, the SNB-1::GFPmarker was localized to dendritic endings as well asthe axonal processes (Figure 2B; see Table S1 in theSupplemental Data available online). A similar pheno-type was observed in lrk-1(km41) mutants (Table S1).For a control, we examined animals expressing theflp-13p::gfp transgene, in which GFP is expressedfrom the flp-13 promoter and found no morphologicalneural abnormalities in lrk-1 mutants (Figures S1A andS1B). Furthermore, the lrk-1 mutation did not affectexpression levels of SNB-1::GFP (Figure 2I). Thus,changes in the localization pattern in lrk-1 mutants arenot the result of alterations in the transcription or trans-lation of SNB-1::GFP.

To confirm whether the mutant phenotype is causedby the lrk-1 mutation, we constructed a flp-13p::lrk-1transgene expressing the lrk-1 cDNA from the flp-13

LRK-1 Regulates Polarized Sorting of SV Proteins593

Figure 1. Structure of LRK-1

(A) Comparison of LRK-1 proteins. Schematic diagrams of C. elegans LRK-1 and human LRRK2/PARK8 proteins are shown. Domains are shown

as follows: leucine-rich repeats (LRR), a Roc domain, a COR domain, a MAPKKK-like kinase domain, and WD40 repeats. The sequence alignment

of LRRK2 and LRK-1 around the PARK8 (I2020T) mutation site is shown. Identical and similar residues are highlighted with black and gray

shading, respectively. The arrow indicates the Ile-2020 residue of LRRK2.

(B) Genomic structure of lrk-1. Exons and introns are indicated by boxes and lines, respectively. Bold lines underneath indicate the extent of the

deleted region in each deletion mutant. The structure of the lrk-1N::gfp fusion gene is also shown.

(C) Expression pattern of the lrk-1N::gfp fusion gene. LRK-1N::GFP was expressed in head neurons (left panel, arrow; neuronal cell bodies), the

ventral nerve cord (right panel, filled-in arrowhead), hypodermis (right panel, asterisks), and intestine (right panel, empty arrowhead). Images are

confocal Z series projected into a single plane. The anterior is on the left. Scale bars represent 10 mm.

promoter. In transgenic lrk-1 mutants harboring theflp-13p::lrk-1 transgene, most animals expressed theSNB-1::GFP marker that showed exclusive localizationto the axonal processes, but not to dendritic processes,in head sensory neurons (Table S1). To address thebiological importance of LRK-1 kinase activity, wegenerated LRK-1(K1726A), a kinase-inactive form ofLRK-1, in which Lys-1726 in the ATP-binding domainhas been mutated to Ala. When the lrk-1(K1726A) genewas expressed from the flp-13 promoter in lrk-1mutants, we observed no rescue of SNB-1::GFP misloc-alization (Table S1). These results suggest that thekinase activity of LRK-1 is crucial to its function in deter-mining polarized SNB-1 localization in neurons. In hu-mans, several mutations in the LRRK2/PARK8 genehave been identified in patients with familial Parkinson-ism [1, 2]. We introduced one of these mutations,lrk-1(I1877T), into the lrk-1 transgene (Figure 1A). Thelrk-1(I1877T) mutation was able to rescue the lrk-1 mu-tant phenotype (Table S1). These results suggest thatthe lrk-1(I1877T) mutation may be a gain-of-functionmutation possessing wild-type lrk-1 activity. Consistentwith this interpretation, the LRRK2 mutants associatedwith Parkinson disease possess augmented kinaseactivity [5–8].

To verify that the mislocalization of SNB-1::GFP in lrk-1mutants was not restricted to this particular transgenemarker, we examined the distribution of the sng-1 gene,encoding the C. elegans SV protein synaptogyrin [9],fused to GFP and expressed from the flp-13 promoter.In wild-type animals, this SNG-1::GFP marker was local-ized to the presynaptic regions of adult head neurons

(Figure 2C). However, in lrk-1 mutants, the SNG-1::GFPmarker was mislocalized to dendritic endings(Figure 2D and Table S1). To determine whether endog-enous SV proteins were regulated similarly, we stainedanimals carrying flp-13p::sng-1::gfp with antibodies toSNB-1. Endogenous SNB-1 was not localized in den-drites in wild-type animals (Figures 2E and 2G). In con-trast, in lrk-1 mutants, endogenous SNB-1 accumulatedin the same dendritic endings where SNG-1::GFP accu-mulated (Figures 2F and 2H). To determine whether lrk-1mutants exhibit a general defect in protein sorting, weexamined the localization of a dendritic protein, theodorant receptor ODR-10 [10]. When ODR-10::GFPexpressed from the str-1 promoter, GFP was found tobe localized to the cilia on dendrites of AWB olfactoryneurons (Figure S1C). This localization was not affectedby the lrk-1 mutation (Figure S1D). Taken together, theseresults suggest that LRK-1 specifically controls thelocalization of SV proteins.

We also examined the localization of SNB-1::GFP andSNG-1::GFP in DD motor neurons. At the first-stage (L1)larvae, DD neurons form synapses to ventral body-wallmuscles [11]. In wild-type animals, SNB-1::GFP andSNG-1::GFP markers were localized along the presyn-aptic ventral processes but not in postsynaptic dorsalprocesses in L1 DD neurons (Figures S2A and S2C).However, in lrk-1 mutant L1s, SNB-1::GFP andSNG-1::GFP were localized along not only the ventralDD processes but also the dorsal DD processes (FiguresS2B and S2D; Table S2). In contrast, a functional post-synaptic GABA receptor marker, UNC-49B::GFP [12],was localized only along the ventral cord in wild-type

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Figure 2. The Effect of the lrk-1 Mutation on Localization of SV Proteins

(A–H) Localization of SV proteins in adult amphid sensory neurons. The dendritic endings and axonal processes of these neurons are indicated by

arrowheads and squares, respectively. In lrk-1(km17) mutants, SNB-1::GFP (B), SNG-1::GFP (D), and endogenous SNB-1 (F) were mislocalized to

the dendritic endings of these neurons. Expression of SNB-1::GFP and SNG-1::GFP were also observed in pharyngeal neurons (indicated by

arrows) and some unidentified neurons. Images shown in (A) and (B) are confocal Z series projected into a single plane. The anterior is on the

left for all panels. Scale bars represent 10 mm.

(I) Immunoblotting of SNB-1. Whole extracts were prepared from worms carrying the indicated transgenes. The upper panel shows immunoblot-

ting with SNB-1 antibody. Positions of SNB-1::GFP and endogenous SNB-1 are indicated by arrows. The lower panel shows immunoblotting with

tubulin antibody as a loading control.

L1 animals, and mutations in lrk-1 did not affect thislocalization (Figures S2E and S2F). This suggests thatLRK-1 affects the transport or sorting of SV componentsbut not the formation of functional dorsal synapses inthe DD neurons.

Genetic Interactions of lrk-1 with syd-1, sad-1,

unc-101, and unc-104 with Regard to PolarizedSV Localization

Mutations in the syd-1 and sad-1 genes affect the axon-dendrite polarity of SV proteins [13–15]. The syd-1 and

LRK-1 Regulates Polarized Sorting of SV Proteins595

Figure 3. The Effects of syd-1, sad-1, unc-104, and unc-101 Mutations on SV Localization and lrk-1 Phenotypes

Localization of SNB-1::GFP (A–F) and SNG-1::GFP (G and H) in adult amphid sensory neurons of indicated worms are shown. syd-1 and sad-1

mutants showed SNB-1::GFP puncta in dendritic processes (indicated by yellow arrows). The unc-104 mutation induced SNB-1::GFP retention in

cell bodies (indicated by bold arrows). Axonal processes and dendritic endings are indicated by squares and arrowheads, respectively. All

images are confocal Z series projected into a single plane. The anterior is on the left. Scale bars represent 10 mm.

sad-1 genes encode a Rho GAP-like molecule and a ser-ine/threonine protein kinase, respectively. To determinewhether LRK-1, SYD-1, and SAD-1 function in the samepathway to control neuronal polarity, we compared thephenotypes of lrk-1 mutants with those of syd-1 andsad-1 mutants. When the flp-13p::snb-1::gfp transgenewas expressed in syd-1(ju82) or sad-1(ky289) animals,the SNB-1::GFP marker was found in both axonal anddendritic processes of adult head sensory neurons.However, SNB-1::GFP in these mutants did not accumu-late in the dendritic endings (Figures 3A and 3B). In L1DD neurons, syd-1 and sad-1 mutations resulted in theappearance of SNB-1::GFP punctate along the dorsal

processes (Figures S3C and S3D). These mutationsalso caused mislocalization of UNC-49B::GFP in L1DDs [13, 15], whereas the lrk-1 mutation did not affectUNC-49B::GFP localization (Figure S2F). Consistentwith this observation, the syd-1 and sad-1 mutationsenhanced dorsal mislocalization of SNB-1::GFP in lrk-1mutant L1s (Figures S3E and S3F). These results sug-gest that LRK-1 regulates axon-dendrite polarity ina manner different from SYD-1 or SAD-1.

In C. elegans, transport of SV components from thecell body to axonal termini requires the unc-104 gene,which encodes the C. elegans homolog of KIF1A kinesin[16, 17]. In unc-104(e1265) mutants, SNB-1::GFP was

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Figure 4. Function and Localization of LRK-1

(A) Time-lapse microscopy of SNB-1::GFP. Time-lapse images of SNB-1::GFP fluorescence around the dendritic tip of head sensory neurons in

unc-104(e1265) mutants (left panels) and lrk-1(km17); unc-104(e1265) double mutants (right panels) are shown. Each photo was taken at 1.2 or

1.5 s intervals. Closed arrowheads indicate accumulated SNB-1::GFP in dendritic endings, and open arrowheads indicate the corresponding

sites. SNB-1::GFP vesicles are indicated by arrows. The anterior is on the left. Scale bars represent 5 mm.

(B) Localization of LRK-1. LRK-1::VENUS and Golgi-marker MIG-23::CFP were expressed under the flp-13 promoter in head neurons. The

merged image is shown in the right panel. Unlike vertebrate cells that contain one large juxtanuclear Golgi stack, most invertebrate cells

such as those in C. elegans instead contain a number of small Golgi stacks per cell, with the individual spots dispersed throughout the cytoplasm.

The scale bar represents 5 mm.

(C) Schematic model of LRK-1 function in neurons.

LRK-1 Regulates Polarized Sorting of SV Proteins597

primarily restricted to cell bodies of adult head sensoryneurons (Figure 3C) and L1 DD neurons (Figure S2G). Bycontrast, in lrk-1(km17); unc-104(e1265) double mu-tants, SNB-1::GFP was greatly diminished in the axonalprocesses but was still mislocalized to dendritic endingsof head sensory neurons (Figure 3D and Table S3) andalong the dorsal processes of L1 DD neurons(Figure S2H). Thus, the mislocalization of SV proteinsto dendritic processes observed in lrk-1 mutants is notmediated by UNC-104 kinesin.

To assess how SNB-1::GFP might be transported intodendrites, we assayed the movement of SNB-1::GFP inhead sensory neurons of lrk-1(km17);unc-104(e1265)double mutants by using time-lapse fluorescencemicroscopy. Rapid transport of SNB-1::GFP from thecell body to dendritic endings was not observed inunc-104 mutants (Figure 4A, left). However, we foundthat SNB-1::GFP was capable of rapid movement towardthe dendritic endings in lrk-1;unc-104 double mutants(Figure 4A, right). The speed of vesicle movement ranged0.4–3.6 mm/s. Anterograde vesicles moved at a meanrate of 1.8 6 0.7 mm/s (n = 16). Thus, the rate of UNC-104-independent SNB-1::GFP movement to dendriteswas comparable to motor-protein-driven transport(0.5–5.0 mm/s) [18]. These results suggest that in the ab-sence of LRK-1, SV proteins are transported to dendritesby another transport system, which is not usually usedfor the transport of SV proteins in wild-type animals.

The unc-101 gene, which encodes a m1 subunit of theAP-1 clathrin adaptor complex, is required for the trans-port of odorant receptors but not of SV proteins in thesensory neurons of worms [19]. In unc-101(m1) mutants,SNB-1::GFP showed normal polarized localization inadult head sensory neurons (Figure 3E) and L1 DDneurons (Figure S2I). In lrk-1(km17) unc-101(m1) doublemutants, SNB-1::GFP was also not detected in dendriticendings of head sensory neurons (Figure 3F and TableS3) or dorsal processes of L1 DD neurons (Figure S2Jand Table S2), but it was normally localized in axonalprocesses of these neurons. Similarly, in lrk-1 unc-101double mutants, SNG-1::GFP was not mislocalized todendritic endings in head sensory neurons (Figure 3Hand Table S1). When pJL2 plasmid containing theunc-101 gene [20] was reintroduced into lrk-1 unc-101double mutants, the SNB-1::GFP marker was mislocal-ized in dendritic endings of head sensory neurons (TableS3). Thus, UNC-101 contributes to the transport of SVproteins to dendrites in lrk-1 mutants.

We next determined the intracellular localization ofLRK-1 by using an lrk-1::venus fusion gene from theflp-13 promoter (flp-13p::lrk-1::venus). Expression ofLRK-1::VENUS was found to rescue the lrk-1 mutantphenotype (Table S1). In lrk-1(km41) mutants carryingthe flp-13p::lrk-1::venus, we detected LRK-1::VENUSonly in cell bodies but not in dendritic or axonal pro-cesses of adult head neurons. LRK-1::VENUS proteinshowed a pattern of punctate cytoplasmic staining,similar to that observed in the Golgi marker MIG-23::CFP[21] (Figure 4B). We found that LRK-1::VENUS colocal-ized extensively with MIG-23::CFP (Figure 4B), suggest-ing that the LRK-1 protein is localized to the Golgi appa-ratus.

Our results raise the possibility that the LRK-1 proteinkinase functions on the trans-Golgi network (TGN) to

exclude SV proteins from the dendrite-specific transportmechanism mediated by the AP-1 clathrin adaptorcomplex (Figure 4C). The loss of LRK-1 causes SV pro-teins to enter the UNC-101-dependent vesicle-sortingpathway, which delivers cargos to the dendritic mem-brane. Thus, LRK-1 may control the selectivity of AP-1for specific cargos so that SV proteins can be excluded.In humans, mutations in LRRK2/PARK8 cause familialParkinsonism [1, 2]. Several other genes are also knownto be involved in Parkinson’s disease, and these includegenes for synphilin-1 and a-synuclein, which are knownas SV-binding proteins and are also enriched in Lewybodies, pathogenic structures in the brains associatedwith Parkinson’s disease [22, 23]. Thus, we may specu-late that LRK-1/LRRK2 proteins regulate the properlocalization of SV proteins in both C. elegans andhumans. Further analysis of LRK-1 function will beimportant for understanding the relationship betweenLRK-1/LRRK2 and SV proteins in neurons.

Supplemental Data

Supplemental Data include Experimental Procedures, three figures,

and four tables and are available with this article online at http://

www.current-biology.com/cgi/content/full/17/7/592/DC1/.

Acknowledgments

We thank C. Bargmann, A. Coulson, A. Fire, Y. Kohara, T. Ishihara, C.

Li, M. Nonet, P. Sternberg, and Caenorhabditis Genetic Center for

materials. J.Y.M. was an associate and Y.J. is an investigator of

the Howard Hughes Medical Institute. This research was supported

by grants from Solution-Oriented Research for Science and Tech-

nology and Ministry of Education, Culture, and Science of Japan

(to K.M., and N.H.).

Received: October 7, 2006

Revised: January 24, 2007

Accepted: January 29, 2007

Published online: March 8, 2007

References

1. Paisan-Ruiz, C., Jain, S., Evans, E.W., Gilks, W.P., Simon, J., van

der Brug, M., Lopez de Munain, A., Aparicio, S., Gil, A.M., Khan,

N., et al. (2004). Cloning of the gene containing mutations that

cause PARK8-linked Parkinson’s disease. Neuron 44, 595–600.

2. Zimprich, A., Biskup, S., Leitner, P., Lichtner, P., Farrer, M., Lin-

coln, S., Kachergus, J., Hulihan, M., Uitti, R.J., Calne, D.B., et al.

(2004). Mutations in LRRK2 cause autosomal-dominant parkin-

sonism with pleomorphic pathology. Neuron 44, 601–607.

3. Nonet, M.L. (1999). Visualization of synaptic specializations in

live C. elegans with synaptic vesicle protein-GFP fusions.

J. Neurosci. Methods 89, 33–40.

4. Kim, K., and Li, C. (2004). Expression and regulation of an

FMRFamide-related neuropeptide gene family in Caenorhabdi-

tis elegans. J. Comp. Neurol. 475, 540–550.

5. West, A.B., Moore, D.J., Biskup, S., Bugayenko, A., Smith, W.W.,

Ross, C.A., Dawson, V.L., and Dawson, T.M. (2005). Parkinson’s

disease-associated mutations in leucine-rich repeat kinase 2

augment kinase activity. Proc. Natl. Acad. Sci. USA 102,

16842–16847.

6. Gloeckner, C.J., Kinkl, N., Schumacher, A., Braun, R.J., O’Neill,

E., Meitinger, T., Kolch, W., Prokisch, H., and Ueffing, M.

(2006). The Parkinson disease causing LRRK2 mutation I2020T

is associated with increased kinase activity. Hum. Mol. Genet.

15, 223–232.

7. Greggio, E., Jain, S., Kingsbury, A., Bandopadhyay, R., Lewis,

P., Kaganovich, A., van der Brug, M.P., Beilina, A., Blackinton,

J., Thomas, K.J., et al. (2006). Kinase activity is required for

the toxic effects of mutant LRRK2/dardarin. Neurobiol. Dis. 23,

329–341.

Current Biology598

8. Smith, W.W., Pei, Z., Jiang, H., Dawson, V.L., Dawson, T.M., and

Ross, C.A. (2006). Kinase activity of mutant LRRK2 mediates

neuronal toxicity. Nat. Neurosci. 9, 1231–1233.

9. Zhao, H., and Nonet, M.L. (2001). A conserved mechanism of

synaptogyrin localization. Mol. Biol. Cell 12, 2275–2289.

10. Sengupta, P., Chou, J.H., and Bargmann, C.I. (1996). odr-10

encodes a seven transmembrane domain olfactory receptor

required for responses to the odorant diacetyl. Cell 84, 899–909.

11. White, J.G., Southgate, E., Thomson, J.N., and Brenner, S.

(1986). The structure of the nervous system of the nematode

Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol.

Sci. 314, 1–340.

12. Byrd, D.T., Kawasaki, M., Walcoff, M., Hisamoto, N., Matsu-

moto, K., and Jin, Y. (2001). UNC-16, a JNK-signaling scaffold

protein, regulates vesicle transport in C. elegans. Neuron 32,

787–800.

13. Hallam, S.J., Goncharov, A., McEwen, J., Baran, R., and Jin, Y.

(2002). SYD-1, a presynaptic protein with PDZ, C2 and rho-

GAP-like domains, specifies axon identity in C. elegans. Nat.

Neurosci. 5, 1137–1146.

14. Crump, J.G., Zhen, M., Jin, Y., and Bargmann, C.I. (2001). The

SAD-1 kinase regulates presynaptic vesicle clustering and

axon termination. Neuron 29, 115–129.

15. Hung, W., Hwang, C., Po, M.D., and Zhen, M. (2007). Neuronal

polarity is regulated by a direct interaction between a scaffolding

protein, Neurabin, and a presynaptic SAD-1 kinase in Caeno-

rhabditis elegans. Development 134, 237–249.

16. Otsuka, A.J., Jeyaprakash, A., Garcia-Anoveros, J., Tang, L.Z.,

Fisk, G., Hartshorne, T., Franco, R., and Born, T. (1991). The C.

elegans unc-104 gene encodes a putative kinesin heavy chain-

like protein. Neuron 6, 113–122.

17. Hall, D.H., and Hedgecock, E.M. (1991). Kinesin-related gene

unc-104 is required for axonal transport of synaptic vesicles in

C. elegans. Cell 65, 837–847.

18. Hirokawa, N. (1998). Kinesin and dynein superfamily proteins

and the mechanism of organelle transport. Science 279, 519–

526.

19. Dwyer, N.D., Adler, C.E., Crump, J.G., L’Etoile, N.D., and Barg-

mann, C.I. (2001). Polarized dendritic transport and the AP-1

m1 clathrin adaptor UNC-101 localize odorant receptors to olfac-

tory cilia. Neuron 31, 277–287.

20. Lee, J., Jongeward, G.D., and Sternberg, P.W. (1994). unc-101,

a gene required for many aspects of Caenorhabditis elegans de-

velopment and behavior, encodes a clathrin-associated protein.

Genes Dev. 8, 60–73.

21. Nishiwaki, K., Kubota, Y., Chigira, Y., Roy, S.K., Suzuki, M.,

Schvarzstein, M., Jigami, Y., Hisamoto, N., and Matsumoto, K.

(2004). An NDPase links ADAM protease glycosylation with

organ morphogenesis in C. elegans. Nat. Cell Biol. 6, 31–37.

22. Wakabayashi, K., Engelender, S., Yoshimoto, M., Tsuji, S., Ross,

C.A., and Takahashi, H. (2000). Synphilin-1 is present in Lewy

bodies in Parkinson’s disease. Ann. Neurol. 47, 521–523.

23. Spillantini, M.G., Schmidt, M.L., Lee, V.M., Trojanowski, J.Q.,

Jakes, R., and Goedert, M. (1997). a-synuclein in Lewy bodies.

Nature 388, 839–840.