一般演題（ポスター）

一般演題（ポスター） Neurite Outgrowth・Network Formation

3P-01 Anti-phosho-GAP-43 pSer96 antibody as a novel molecular marker for axonal growth and regeneration Okada Masayasu^1,2，Kawasaki Asami^2,3，Takeuchi Kosei^2,4，Tamada Atsushi^2,3，Nakatsu Fubito²，Fujii Yukihiko¹，Igarashi Michihiro^2,3 ¹Department of Neurosurgery, Brain Research Institute, Niigata University，²Department of Neurochemistry and Molecular Cell Biology, Niigata University, Graduate School of Medical and Dental Sciences，³Center for Transdisciplinary Research, Niigata University，⁴Department of Cell Biology, Aichi medical University GAP-43 is one of the good molecular markers for axonal regeneration after injury. We performed the phosphoproteomics using the growth cone fraction and identified several novel phosphorylation sites of GAP-43. We established the specific antibodies against these sites（Igarashi et al., in preparation）. To evaluate one of them, the anti-phospho-GAP-43 pSer96 antibody, as a marker of the axonal regeneration in vivo, we performed the following experiments. We performed a standard protocol for the sciatic nerve axon injury using adult C57B6N mice（J Neurosci Methods 227（2014））. We evaluated crush nerves（on day 3）and uninjured control nerves, by western blotting and immunohistochemistry using pSer96-GAP-43 antibody. As for the regeneration assay, we adopted the confocal micrographs along the longitudinal nerve section and used Regeneration Index, which is designated by the measured distance, from the crush site（point A）to the site at which pSer96-GAP-43 intensity level is half of that at point A（Shin J.E. et al. Neuron 74（2012））. The western blots showed the high intensity of pSer96-GAP-43 staining in the crushed nerve（day 3）and the very low intensity in the control nerve. Immunohistochemistry revealed the co-localization of pSer96-GAP-43 with TUJ-1 at the injury site and the very low staining of pSer96-GAP-43 in the control nerve. The Regeneration Index with pSer96-GAP-43 was almost equivalent to the result using anti-SCG10 antibody, a marker of sensory axon regeneration at an acute phase. Taken together from these results, we concluded the excellent utility of pSer96-GAP-43 antibody in vivo as a regeneration marker.		3P-02 JNK-mediated phosphorylation of GAP-43 promotes axonal growth Kawasaki Asami^1,2，Kobayashi Daiki¹，Okada Masayasu³，Takeichi Kosei⁴，Igarashi Michihiro^1,2 ¹Dept. of Neurochem. and Mol. Cell Biol., Niigata Univ.，²Trans-Disciplinary Res. Prog., Niigata Univ.，³Dept. of Cell. Neurobiol., Brain Res. Inst., Niigata Univ.，⁴Dept. of Med., Aichi Med. Univ. Growth-associated Protein-43 kDa（GAP-43）is involved in the mechanisms regulating the growth of neuronal processes during development and axon regeneration. However, its role for the molecular signaling is poorly understood. Recently, we had performed a quantity phosphorproteomic analysis of axonal growth cones and determined more than 1,000 phosphorylation sites. We identified some novel phosphorylation sites of GAP-43, which are extensively highly phosphorylated in in vivo. By using specific antibodies of phospho-GAP-43 at these sites, we confirmed that these sites were highly phosphorylated not only in the developing brain but also in the regrowing axons of the spinal cord. In in silico and in vitro examinations, we identified c-Jun N-terminal kinases（JNKs）was a major kinase responsible for these sites. Inactivation of a phosphorylation site by a point mutation delayed axonal growth in vitro. These results suggest that JNK-dependent phosphorylation of GAP-43 is one of the important signaling involved in axonal generation and regeneration in vivo. We are now investigating a role of JNK-dependent GAP-43 phosphorylation in the course of neuronal wiring.

3P-03 Neurite outgrowth and bipolarization in PC12 cells and cerebral cortical neurons induced by a low concentration of bisphenol A Aoyama Hiroki¹，Fujieda Satoshi²，Genko Soichiro¹，Matsuura Kumi²，Mizui Toshiyuki³，Kojima Masami³，Shimoke Koji^1,2 ¹Grad. Sch. Sci. Tech., Kansai Univ.，²Dept. Life Sci. Biotech., Fac. Chem. Mat. Bioengin., Kansai Univ.，³National Institute of AIST, Health Research Institute Endocrine disrupting chemicals（EDCs）are known to exhibit hormone-like effects and inhibit specific nuclear receptors, such as endogenous hormone receptors that maintain homeostasis in many creatures. Recently, we found that some EDCs induce apoptosis, and these EDCs are referred to as“apoptogens”. One of them, bisphenol A（BPA）, is widely used in the production of plastics, and it has been reported that BPA is harmful to the central nervous system. In a recent study, we showed that BPA induces neurite outgrowth in PC12 cells at a relatively low concentration. In the current study, we demonstrated that BPA also induces PC12 cells and cerebral cortical neurons to form bipolar neuronal cells. Specifically, we compared cultured PC12 cells treated with 60 μM BPA and 100 ng/ml NGF. As a result, we found that the neurites induced by BPA exhibited fewer neurite branches compared with those induced by NGF, and that BPA also induced cell body bipolarization. The neurite outgrowth was inhibited by treatment with an estrogen receptor antagonist（ICI182780）. Furthermore, we also demonstrated that neurite outgrowth could be induced in cerebral cortical neurons derived from fetal rat brain（E17-18）by a low concentration of BPA. Thus, BPA may seriously affect neuronal differentiation by changing these cells into bipolar neuronal cells. We will further investigate the specific molecules related to estrogen receptor（ER）signaling that are involved in the elongation of short neurites in PC12 cells and cerebral cortical neurons. As this phenomenon may contribute to pathophysiological development, a detailed understanding of the molecular mechanism（s）, including ERs, that are affected by BPA, will be essential to prevent neurite outgrowth or bipolarization.		3P-04 Activation of RhoA/Rho-kinase by CaMKI-mediated phosphorylation of GEF-H1 regulates neuronal polarization Takano Tetsuya，Nakamuta Shinichi，Wu Mengya，Ishizawa Naruki，Xu Chundi，Namba Takashi，Amano Mutsuki，Kaibuchi Kozo Dept. of Cell Pharmacology. Med., Univ. of Nagoya Neurons are highly polarized cells with structurally and functionally different processes, an axon and several dendrites. One of minor neurites begins to extend rapidly and differentiates into the axon. During the axonal outgrowth, minor neurites consistently grow and retract to prevent multiple axon formation, thereby maintaining neuronal polarity. However, the molecular mechanisms that maintain neuronal polarity remain largely unknown. Here, we found that retrograde long-range Ca2+ signaling regulates the maintenance of neuronal polarity by increasing RhoA/Rho-kinase activity thorough GEF-H1/Lfc, a RhoA-specific guanine nucleotide exchange factor（GEF）, in a Ca2+/calmodulin-dependent protein kinase I（CaMKI）-dependent manner. The minor neurites were retracted by local application of Ca2+ ionophore to axon terminal, probably through the propagation of Ca2+ wave to soma and/or minor neurite. Local application of Rho-kinase inhibitor to minor neurite induced rapid elongation and subsequent multiple axon formation. Moreover, we found that CaMKI phosphorylated GEF-H1 at Thr103. The phosphorylation of GEF-H1 at Thr103 by CaMKI significantly increased its GEF activity. The phosphomimic mutant of GEF-H1（T103E）impaired neuronal polarization. Taken together, these results suggest that the long-range Ca2+ signaling from axon terminal activates CaMKI and thereby phosphorylates GEF-H1 at other minor neurites. This phosphorylation leads to increase the GEF-H1 activity and in turn to stimulate the RhoA-Rho kinase activity to prevent the formation of multiple axons.

3P-05 Function and expression of the mouse RasGEF1 family proteins Hosaka Kenta，Shinoda Yo，Furuichi Teiichi Fac of Sci and Tech, Tokyo Univ of Sci The mammalian RasGEF1 is known as one of the protein families containing RasGEF domain which is guanine nucleotide exchange factors for Ras-like small GTPases. RasGEF1 protein family consists of 3 distinct types 1a, 1b, and 1c. It was reported that RasGEF1a and 1b activate a member of the Ras protein family Rap2 which is known to synaptic function, modulate cell adhesion and cell morphology. RasGEF1b was shown to interact with a small GTPase Cdc42 on the midbody during cell division. In terms of gene expression, RasGEF1a is predominantly expressed in the central nervous system in human, and RasGEF1b is expressed in midbrain and hindbrain in zebrafish. However, detailed information on the RasGEF1 family is largely unknown. In this study, we cloned their cDNAs and analyzed expression in mouse brain and effect of their over-expression in culture cells. RT-PCR analysis of mouse tissues showed RasGEF1c was predominantly expressed in brain. Microarray analysis during mouse cerebellar postnatal development showed that RasGEF1a was up-regulated, whereas RasGEF1b and 1c were down-regulated. According to in situ hybridization data of Allen Brain Atlas, mRNA of each RasGEF1 type displays widespread but differential distribution patterns in mouse brain. Our preliminary data also suggested neurite outgrowth and morphological changes of cultured cells exogenously over-expressed RasGEF1 proteins. Together, these data suggest that each member of the RasGEF1 family may play a role in cell signaling during specific developmental stages and in distinct brain regions.		3P-06 Lemur Kinase 1A（LMTK1A）may coordinate membrane and cytoskeletal dynamics in neurite outgrowth. Sharma Govinda¹，Tsutsumi Koji¹，Asada Akiko¹，Saito Taro¹，Tomomura Mineko² ¹Laboratory of Molecular Neuroscience, Department of Biology, Graduate School of Science, Tokyo Metropolitan University，²Meikai Pharmaco-Medical Laboratory, Meikai University, School of Dentistry Lemur kinase 1A（LMTK1A）, a substrate of CDK5/p35, is a Ser/Thr kinase highly expressed in mammalian brain. LMTK1A consists of an N-terminal kinase domain and long C-terminal tail. It is palmitoylated at three cysteine residues in the N-terminal region that anchors it to recycling endosomes. We have previously reported that LMTK1A inhibits neurite outgrowth via modulation of Rab11A, a small GTPase, which regulates recycling endosome traffic. However, it is unknown yet how the kinase activity is involved in neurite outgrowth. Neurite outgrowth is complicated processes involving both cytoskeletal dynamics and membrane transport, but it is not known how they are coordinated. In this study, we examined the role of kinase activity of LMTK1A in its interaction with the cytoskeletons, especially microtubules（MTs）and actin filaments. We found that wild type（wt）LMTK1A was predominantly localized in pericentrosomal area containing MTOC, while kinase negative（kn）mutant of LMTK1A is distributed evenly throughout the whole cytoplasm. Further, in the neurite tips wtLMTK1A was accumulated at the tip of MTs and did not invade into the cortical actin-rich region. In contrast, knLMTK1A was found in the actin-rich cortical region as well as MT-rich cytoplasm. In addition, the pericentrosomal localization of LMTK1A was abolished when MTs were destabilized with nocodazole, but when nocodazole was washed out and MTs regrew, LMTK1A re-localized to the pericentrosomal area. Although it is not yet clear how LMTK1A affects organization and dynamics of MTs, these results suggest that LMTK1A regulates a critical step of membrane transport from MTs to the cortical actin in neurite tip, which is necessary for neurite outgrowth.

3P-07 Role of N-glycans to a function of a trans-membrane protein, seizure-related gene 6（sez-6） Hidaka Chiharu，Mitsui Shinichi Department of Rehabilitation Sciences, Gunma University Graduate School of Health Sciences In the developing central nerves system, trans-membrane proteins play important roles in various steps for the construction of the neuronal circuit including the cell-migration, differentiation, axon elongation, dendritic formation and synaptogenesis. It is well known that trans-membrane proteins are modified by addition of N-glycan. N-linked glycan regulates functions of proteins, since it contributes to folding and stability of proteins. Seizure-related gene 6（sez-6）is a trans-membrane protein expressed in cerebral cortex and hippocampus, modulates dendritic branching. Sez-6 contains eleven putative N-glycosylation sites. The role of N-glycans on sez-6 is still obscure. To understand the function of N-glycans on sez-6, we investigated neuro2a cells overexpressing sez-6 mutants. Eleven N-glycosylation sites of sez-6 are divided to three clusters which we termed sugar chain（SC）1-3, SC4-7, SC8-11. The mutants we prepared lacked one, two or all N-glycosylation clusters. Mutants（sez-6 SC1-3, SC8-11）having one N-glycosylation cluster at the position as well as a mutant lacking all clusters（sez-6 Δ1-11）were transported to the cell membrane but were not distributed to fine processes. On the contrary, sez-6 SC4-7 mutant and mutants lacking one N-glycosylation cluster（sez-6 ΔSC1-3, ΔSC4-7, ΔSC8-11）were well distributed on the cell membrane like wild type sez-6. Among mutants behaving like wild type sez-6, sez-6 ΔSC1-3 and ΔSC4-7 reduced neurite formation. Interestingly, sez-6 ΔSC4-7 mutant had no effects on the formation of filopodia-like protrusions, which were induced by the overexpression of other mutants and wild type sez-6. Ours results suggest that N-glycans on sez-6 modulate cell morphology by maintaining proper distribution of sez-6 protein on the cell membrane.		3P-08 Deletion of FILIP influenced the development of peripheral nerve Yagi Hideshi¹，Kanda Hirosato²，Noguchi Koichi²，Sato Makoto^3,4,5 ¹Department of Anatomy and Cell Biology, Hyogo College of Medicine，²Department of Anatomy and Neuroscience, Hyogo College of Medicine，³Research Center for Child Mental Development, University of Fukui，⁴United Graduate School of Child Development, Osaka Univ, Kanazawa Univ, Hamamatsu Univ Sch of Med, Chiba Univ and Univ of Fukui，⁵Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University FILIP（filamin A interacting protein）controls neuronal cell migration during the cortical development. We recently reported that FILIP controls neuronal cell morphology via binding to non-muscle myosin IIb. We here found that FILIP was involved in the development of the sensory system. As we observed the expression of FILIP in the neurons of the dorsal root ganglia throughout the development, we investigated the development of the dorsal root ganglia of the FILIP knockout mice. We found that there were mild abnormalities in the neuronal density in the developing dorsal root ganglia of the FILIP knockout mice at the embryonic age. As we suspected developmental delay of the sensory system in the FILIP knockout mice, we studied the innervation of the peripheral nerve to the skin of the FILIP knockout mice using the whole-mount immunohistochemical method. We observed that the delayed innervation of the peripheral nerve to the skin of the FILIP knockout mice. As FILIP controls intracellular distribution of the non-muscle type myosin IIb that plays an important role in the axon elongation, we considered that the deletion of FILIP influenced the elongation of the neurites of somatosensory neurons via myosin IIb.

3P-09 Visualizing single-neuron identity defined by Pcdh-β cluster in mouse brain Kaneko Ryosuke¹，Abe Manabu²，Watanabe Masahiko⁴，Sakimura Kenji²，Yanagawa Yuchio¹，Yagi Takeshi³ ¹Gunma Univ. Grad. Sch. Med.，²Brain Res Inst, Niigata Univ.，³Grad. Sch. Front. Biosci., Osaka Univ.，⁴Grad. Sch. Med., Hokkaido Univ. The brain contains a huge number of neurons that have diverse characteristics participating in discrimination between individual neurons. It has been speculated that clustered protocadherins（Pcdhs）, which encode cadherin-related transmembrane proteins as gene clusters in vertebrate genome, could provide these kinds of neuronal identity. The murine Pcdhs are further classified into three subfamilies：Pcdh-α（14 genes）, Pcdh-β（22 genes）, and Pcdh-γ（22 genes）. Their loss of function in mice revealed that the Pcdhs play important roles in neuronal survival, axonal projection, synaptic connectivity, and several brain functions including learning and memory. As revealed by histological examinations and single-cell RT-PCR, the Pcdhs show the scattered expression in each cerebellar Purkinje cell. The scattered expressions of the Pcdhs will provide a potential neuronal identity at the single-neuron level. The involvement of the scattered Pcdh expression in neural circuit formation has been inferred on the basis of several genetic analyses including loss of Pcdh-γ and loss of gene regulators of the Pcdhs（CTCF and Dnmt3b）. However, several key questions remain unanswered. For example, are the Pcdh expressions scattered in other neuron type? Are the Pcdh expressions dynamically changed in a live neuron? Does the Pcdh expression depend on cell-lineage? In order to answer these questions, we generated knock-in mice that harbor cDNA encoding red fluorescent protein, tdTomato, under the control of endogenous Pcdh-β3 promoter. The mice showed scattered tdTomato fluorescence in various neuron types, including cerebellar Purkinje cells, hippocampal CA1 pyramidal cells, dentate gyrus granule cells, cerebellar molecular layer interneurons, etc. The newly developed antibody against tdTomato enables a high signal-to-noise ratio visualization of single-neuron identity. We are currently addressing the key questions about scattered Pcdh expression.		3P-10 Analysis of protein synthesis in growth cones of rat dorsal root ganglion neurons Hoshi Osamu¹，Cho Yuichiro¹，Takei Nobuyuki² ¹Tokyo Medical and Dental University，²Department of Molecular Neurobiology, Brain Research Institute, Niigata University Although the concept of local translation in neurons is widely accepted, there is a debate about whether axonal translation occurs. Herein, we analyzed the presence of ribosomal proteins in the growth cones of rat dorsal root ganglion（DRG）neurons, by immunofluorescence analysis. Actual protein synthesis was monitored by the surface sensing of translation（SUnSET）method. Structural analysis was performed using atomic force microscopy（AFM）. DRG neurons were prepared from embryonic rats and dissociated, then resuspended in culture medium and plated onto dishes. They were maintained in DMEM containing CPT-cAMP to facilitate axon elongation and growth cone formation for 48h. Neurons were stimulated with brain-derived neurotrophic factors（BDNF）for 30min to induce translational activation under the presence of puromycin. Low dose puromycin binds to elongated peptide chain, thus newly synthesized proteins coupled with puromycin can be detected by anti-puromycin. After AFM observation, specimens were labeled with Alexa 488 phalloidin for actin filament staining, followed by anti-ribosomal protein P0/P1/P2 antibody. Some specimens were labeled with anti-puromycin antibody and anti-P-eEF2（Phosphorylated eukaryotic elongation factor 2）. Immunofluorescence images revealed that actin filaments were distributed in the peripheral region and in the filopodia. The positive regions of ribosomal protein P0/P1/P2 were closely related to the distribution of actin filaments. AFM images showed that high regions of DRG tended to be rich in actin filaments and ribosomal protein P0/P1/P2, compared with low regions of DRG. BDNF decreased the phosphorylation of eEF2, indicating enhancement of translation in growth cones. Indeed, BDNF increased puromycin signaling, which suggests increased protein synthesis in growth cones. These results are discussed in relation to locally-synthesized proteins and are related to the three-dimensional structure of DRG.

3P-11 Drebrin stabilizes CaMKIIβ in core region but not in postsynaptic density of dendritic spine Yamazaki Hiroyuki，Shirao Tomoaki Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine Dendritic spines are actin-rich small protrusions that contain postsynaptic components of excitatory synapse. Many actin-binding proteins have been identified as spine-resident protein, and they regulate actin-cytoskeleton through diverse processes. Drebrin is a major F-actin binding protein in neurons, and is localized in the center of dendritic spines. Drebrin regulates dendritic spine morphogenesis and spine targeting of synaptic proteins such as spikar, PSD-95 and NMDA receptors. Moreover, drebrin is involved in neurological diseases（eg., Alzheimer’s disease and schizophrenia）. Although increasing evidences show that drebrin plays pivotal roles in neurons, how drebrin interacts with other proteins in spines is much less known. In this study, we isolated CaMKIIβ as a drebrin-binding protein by yeast two-hybrid screen and investigated the interaction of drebrin-CaMKIIβ in dendritic spines. CaMKIIβ is localized in dendritic spines more than in dendritic shaft. However, drebrin knockdown（KD）caused diffuse localization of CaMKIIβ in dendrites, suggesting that drebrin anchors CaMKIIβ in dendritic spines. To analyze drebrin-dependence of CaMKIIβ stability in dendritic spine, we performed fluorescence recovery after photobleaching（FRAP）experiments on individual dendritic spines. We calculated the stable fraction from the time-series of fluorescence intensity of GFP-CaMKIIβ before and after photobleaching. The stable fraction of GFP-CaMKIIβ in drebrin-KD neurons was greater than that of control neurons. In addition, NMDA receptor stimulation increased the stable fraction of CaMKIIβ in parallel with drebrin-dislocation from dendritic spines. These results suggest that drebin-loss increases the stable fraction of CaMKIIβ in dendritic spines. Therefore, we think that drebrin-independent stable pool became dominant in drebrin-KD neurons and synaptic activity regulates the accumulation of drebrin-independent CaMKIIβ in dendritic spines. Taken together, our study suggests that there are two stable pools of CaMKIIβ in spines, drebrin-dependent and drebrin-independent pools.		3P-12 Structural basis for cargo binding and autoinhibition of retrograde transport adaptor Bicaudal D1 Yoshikane Asuka¹，Terawaki Shin-ichi¹，Higuchi Yoshiki²，Wakamatsu Kaori¹ ¹Graduate School of Science and Technology, Gunma University，²Department of Picobiology and Department of Life Science, Graduate School of life Science, University of Hyogo Bicaudal D1（BICD1）mediates the attachment of specific cargo to cytoplasmic dynein. The attachment regulation toward cytoplasmic dynein by BICD1 plays an essential role in the minus end-directed intracellular retrograde transport along microtubules. Dysfunction of cargo sorting by BICD1 to cytoplasmic dynein causes various diseases such as dominant congenital spinal muscular atrophy（DCSMA）. BICD1 possess three α helical coiled coil（CC）regions：an N terminal CC1, a central CC2, and a C terminal CC3 region. The N terminal region of BICD1, containing CC1 and a portion of CC2, associates with cytoplasmic dynein, whereas the BICD1 CC3 has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein2（RanBP2）, and inhibiting the associating with cytoplasmic dynein by binding to the CC1. The cargo binding of CC3 promotes association of cytoplasmic dynein by inducing the release of CC1 from CC3. However, the molecular mechanisms, by which the CC3 binds cargo factors and CC1, are unknown. Here, we report the X-ray crystallographic structural analysis of CC3 and the mutational binding experiments with Rab6, RanBP2, and CC1. Firstly, we succeed in the crystallization and the structural determination of CC3 by X-ray crystallographic analysis with a resoution of 1.50 ?. The structure revealed that CC3 forms a parallel homodimeric coiled coil with leucine zipper-like heptad repeat sequence. Next, we attempted to determine the binding site for cargo factors on CC3. The mutational binding study based on the CC3 structure indicated that CC3 possesses the binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1 binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD1 proteins on the dynein-dependent intracellular retrograde transport.

3P-13 The contribution of the di-leucine motif in p35 to determine the distribution difference between neuronal cyclin-dependent kinase 5（Cdk5）activators p35 and p39 Asada Akiko，Saito Taro，Hisanaga Shin-ichi Dept. Biol. Sci., Tokyo Metro. Univ. Cdk5 is a Proline-directed Ser/Thr kinase regulates a variety of neuronal activities. The Cdk5 activator p35 or p39 also determine the distribution of Cdk5. p35 and p39 are isoforms with a high homology in the C-terminal Cdk5-activating domain but low homology at N-terminal except for the N-myristoylation consensus sequence and the Lys cluster. Previously, we have reported that myristoylation and the Lys cluster are important in their cellular distribution. Though both p35 and p39 localizes at the perinuclear region and plasma membrane, p35 distribute more in perinuclear region than plasma membrane and p39 distribute in contrary pattern to p35. Because myristoylated proteins are found in various intracellular membrane-bound compartments, it is unlikely that only myristoylation determines the specific membrane compartments. To answer this question, we interested in the di-leucine motif, which was found as an ER retention motif at first, and has been also considered to contribute the sorting of protein. p35 has this motif, but p39 does not. To search the effect of this motif, we constructed the deletion mutant of p35 di-leucine motif, p35dCT, and chimera mutant of p39 replacing C-terminal with p35 C-terminal that contained the di-leucine motif. The distribution pattern of p35 dCT in N2A cells exhibited less in perinuclear region and more at plasma membrane than that of WT p35, so to say the intermediate pattern between p35 and p39. The distribution of chimera also exhibited the intermediate pattern between p35 and p39. The di-leucine motif participates to determine the specific membrane localization in the cell, but it is not sufficient. Based on these date, we discussed about the mechanism of determination of distribution of p35 and p39.