Ndel1 palmitoylation: a new mean to regulate cytoplasmic dynein activity
2009; Springer Nature; Volume: 29; Issue: 1 Linguagem: Inglês
10.1038/emboj.2009.325
ISSN1460-2075
AutoresAnat Shmueli, Michal Segal, Tamar Sapir, Ryouhei Tsutsumi, Jun Noritake, Avi Bar, Sivan Sapoznik, Yuko Fukata, Irit Orr, Masaki Fukata, Orly Reiner,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle19 November 2009free access Ndel1 palmitoylation: a new mean to regulate cytoplasmic dynein activity Anat Shmueli Anat Shmueli Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Michal Segal Michal Segal Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Tamar Sapir Tamar Sapir Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ryouhei Tsutsumi Ryouhei Tsutsumi Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan Search for more papers by this author Jun Noritake Jun Noritake Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan Search for more papers by this author Avi Bar Avi Bar Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Sivan Sapoznik Sivan Sapoznik Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yuko Fukata Yuko Fukata Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan PRESTO, Japan Science and Technology Agency, Chiyoda, Tokyo, Japan Search for more papers by this author Irit Orr Irit Orr Biological Services, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Masaki Fukata Masaki Fukata Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan PRESTO, Japan Science and Technology Agency, Chiyoda, Tokyo, Japan Search for more papers by this author Orly Reiner Corresponding Author Orly Reiner Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Anat Shmueli Anat Shmueli Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Michal Segal Michal Segal Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Tamar Sapir Tamar Sapir Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ryouhei Tsutsumi Ryouhei Tsutsumi Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan Search for more papers by this author Jun Noritake Jun Noritake Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan Search for more papers by this author Avi Bar Avi Bar Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Sivan Sapoznik Sivan Sapoznik Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yuko Fukata Yuko Fukata Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan PRESTO, Japan Science and Technology Agency, Chiyoda, Tokyo, Japan Search for more papers by this author Irit Orr Irit Orr Biological Services, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Masaki Fukata Masaki Fukata Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan PRESTO, Japan Science and Technology Agency, Chiyoda, Tokyo, Japan Search for more papers by this author Orly Reiner Corresponding Author Orly Reiner Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Author Information Anat Shmueli1, Michal Segal1,‡, Tamar Sapir1,‡, Ryouhei Tsutsumi2, Jun Noritake2, Avi Bar1, Sivan Sapoznik1, Yuko Fukata2,3, Irit Orr4, Masaki Fukata2,3 and Orly Reiner 1 1Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel 2Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan 3PRESTO, Japan Science and Technology Agency, Chiyoda, Tokyo, Japan 4Biological Services, The Weizmann Institute of Science, Rehovot, Israel ‡These authors contributed equally to this work *Corresponding author. Department of Molecular Genetics, Weizmann Inst. of Science, The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: +972 8 9342319; Fax: +972 8 9344108; E-mail: [email protected] The EMBO Journal (2010)29:107-119https://doi.org/10.1038/emboj.2009.325 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities, and failure to regulate this activity can result in neuronal migration retardation or neuronal degeneration. The activity of dynein is controlled by the LIS1–Ndel1–Nde1 protein complex that participates in intracellular transport, mitosis, and neuronal migration. These biological processes are subject to tight multilevel modes of regulation. Palmitoylation is a reversible posttranslational lipid modification, which can dynamically regulate protein trafficking. We found that both Ndel1 and Nde1 undergo palmitoylation in vivo and in transfected cells by specific palmitoylation enzymes. Unpalmitoylated Ndel1 interacts better with dynein, whereas the interaction between Nde1 and cytoplasmic dynein is unaffected by palmitoylation. Furthermore, palmitoylated Ndel1 reduced cytoplasmic dynein activity as judged by Golgi distribution, VSVG and short microtubule trafficking, transport of endogenous Ndel1 and LIS1 from neurite tips to the cell body, retrograde trafficking of dynein puncta, and neuronal migration. Our findings indicate, to the best of our knowledge, for the first time that Ndel1 palmitoylation is a new mean for fine-tuning the activity of the retrograde motor cytoplasmic dynein. Introduction Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities ranging from mitosis to long-range neuronal transport. For example, mice lacking cytoplasmic dynein heavy chain exhibit early embryonic lethality (Harada et al, 1998), whereas abnormal neuronal transport is a common theme underlying pathogenesis of neurodegenerative diseases (for reviews, see Bruijn et al, 2004; Chevalier-Larsen and Holzbaur, 2006; Reiner et al, 2006; Stokin and Goldstein, 2006). Dynein, a large multisubunit complex, belongs to one of the two families of microtubule motor proteins (for reviews, see Goldstein and Yang, 2000; Vallee et al, 2004). All dyneins contain the heavy chain, for ATPase and motor activities, and accessory subunits including light intermediate and light chains. The activity of dynein is regulated at multiple levels. For example, specific combinations of dynein isoforms may contribute to cargo specificity (Ha et al, 2008). Additional accessory proteins complexes, such as dynacin, allow dynein to bind a variety of cargoes, regulate dynein motor activity directly, and enhance dynein processivity (for reviews, see Schroer, 2004; Vallee et al, 2004). Finally, accumulating evidence suggest that the LIS1-containing protein complex is also involved in regulating cytoplasmic dynein motor activity. Deletions in the LIS1 gene result in a severe human neuronal migration deficit known as lissencephaly (Reiner et al, 1993; Lo Nigro et al, 1997; Pilz et al, 1998). Protein dosage in this locus is crucial because both decreased and increased LIS1 protein levels affect brain development both in humans and in mice (Reiner et al, 1993; Hirotsune et al, 1998; Cahana et al, 2001; Bi et al, 2009). LIS1 has been found to interact with several subunits of dynein and dynactin (Faulkner et al, 2000; Sasaki et al, 2000; Smith et al, 2000; Tai et al, 2002), as well as with the microtubule plus end-binding protein CLIP-170 (Coquelle et al, 2002), suggesting that it modulates dynein activity in more than one way. LIS1 interacts tightly with the evolutionary conserved NUDE proteins (Efimov and Morris, 2000). In mammals there are two NudE homologs: Nde1 and its related paralog Ndel1. Nde1 interacts with LIS1, several centrosomal proteins, and dynein light and intermediate chains (Feng et al, 2000; Feng and Walsh, 2004; Hirohashi et al, 2006a, 2006b; Stehman et al, 2007). Ndel1 is found in complex with LIS1, and dynein heavy and intermediate chains (Sasaki et al, 2000; Niethammer et al, 2000a). LIS1, Ndel1, and Nde1 all participate in the dynein-mediated processes of intracellular transport (Liu et al, 2000; Sasaki et al, 2000; Smith et al, 2000; Niethammer et al, 2000b; Liang et al, 2004; Yamada et al, 2008), mitosis (Faulkner et al, 2000; Yan et al, 2003; Feng and Walsh, 2004; Tsai et al, 2005; Guo et al, 2006; Liang et al, 2007; Stehman et al, 2007; Vergnolle and Taylor, 2007), and neuronal migration (Hirotsune et al, 1998; Feng et al, 2000; Cahana et al, 2001; Feng and Walsh, 2004; Shu et al, 2004; Sasaki et al, 2005; Tsai et al, 2005, 2007; Grabham et al, 2007). These complex biological processes are subject to tight multilevel modes of regulation, in which reversible posttranslational modifications have an important role. For example, phosphorylation of Ndel1 is required for neuronal migration and function (Niethammer et al, 2000b; Toyo-oka et al, 2003; Taya et al, 2007), as well as for proper cell-cycle progression (Toyo-Oka et al, 2005; Mori et al, 2007). In vitro studies have demonstrated that LIS1 can stimulate the ATPase activity of cytoplasmic dynein (Mesngon et al, 2006; Yamada et al, 2008). Furthermore, LIS1 suppressed the motility of cytoplasmic dynein on microtubules, whereas its interacting protein Ndel1 released the blocking effects of LIS1 (Yamada et al, 2008). In vivo, injection of anti-LIS1 antibodies inhibited the anterograde transport of dynein to the tips of dorsal root ganglia neurons, thus suggesting that LIS1 may affect the activity of the anterograde motor kinesin in relation to dynein transport (Yamada et al, 2008). Taking into consideration that reversible posttranslational modifications are likely to participate in the regulation of dynamic processes, we tested whether any of the LIS1–Ndel1–Nde1 complex proteins undergo palmitoylation. Palmitoylation is a reversible posttranslational modification, which has been shown to have an important role in the regulation of protein trafficking (Linder and Deschenes, 2007), and in the nervous system (Kang et al, 2008; for reviews, see Dunphy and Linder, 1998; Resh, 1999; El-Husseini Ael and Bredt, 2002; Washbourne, 2004). We found that both Ndel1 and Nde1 undergo palmitoylation on a conserved cysteine residue. We further identified the three palmitoylation enzymes DHHC2, DHHC3, and DHHC7, which are involved in this modification. Palmitoylation of Ndel1 reduced its interaction with cytoplasmic dynein resulting in reduced dynein activity measured by functional assays, including Golgi distribution, microtubule transport, ER-to-Golgi transport, transport to neurite tips, retrograde trafficking of dynein puncta in primary neurons, and aberrant pyramidal neuronal migration to the developing cortex. Our results suggest a new mode of regulating the activity of the molecular motor, cytoplasmic dynein. Results Nde1 and Ndel1 are palmitoylated proteins We tested whether Ndel1/Nde1 or LIS1 undergo palmitoylation. S-palmitoylation is a reversible posttranslational modification, which involves a thioester-bound addition of the fatty acid palmitate to cysteine residues (for review, see Linder and Deschenes, 2007). In mammals there are 23 known palmitoylating enzymes called palmitoyl-acyl transferases (PAT) (Fukata et al, 2004) that share in common an Asp-His-His-Cys (DHHC)-cysteine rich domain that is essential for the catalytic activity. The 23 PATs (DHHC 1–23) were transfected to HEK293 cells and assayed (Fukata et al, 2006) for their capability to palmitoylate Ndel1 or Nde1 (Figure 1A and B, Supplementary Figure S1 is a longer exposure of 1B). This assay involves metabolic labelling of tritiated-palmitate to HEK293 cells transfected with the enzymes and the tested proteins. The results indicated that both proteins are palmitoylated, and the enzymes that are capable of palmitoylating Ndel1 and Nde1 are DHHC2, DHHC3, and DHHC7. Minor palmitoylation was noted with DHHC21. LIS1 was not palmitoylated using the same assay (data not shown). We next explored whether Ndel1 and Nde1 are palmitoylated in vivo in primary cortical neurons using a sensitive assay (Drisdel and Green, 2004; Drisdel et al, 2006). This assay involves an exchange of the endogenous palmitate-linked group with a biotin-labelled reagent. This reaction requires hydroxylamine-mediated cleavage of the palmitoyl-thioester bond, followed by a specific labelling with a sulfhydryl-reactive biotinylated reagent that is later identified by avidin-HRP (known as ABE, acyl-biotinyl exchange method). Our results indicate that both Ndel1 and Nde1 are palmitoylated in vivo in mouse cortical neurons (Figure 1C and D). In both cases, avidin–HRP labelled immunoprecipitated only Ndel1 (Figure 1C) or Nde1 (Figure 1D) when hydroxylamine hydrolysed the thioester as a neutral pH base, but not when it was not added. It has been previously demonstrated that a band shift of PSD95 can be noted after the addition of hydroxylamine (Fukata et al, 2004; Figure 1E). We noted a similar band shift of Ndel1 (Figure 1F), thus suggesting that a noticeable proportion of Ndel1 population is palmitoylated in CAD cells. Determination of the half-life of GFP–Ndel1 palmitoylation in an inducible DHHC7 HEK293 cell line was conducted using metabolic labelling of the cells with 17-octadecynoic acid (17-ODYA) overnight, followed by a chase with palmitic acid (Figure 1G and H). The half-life of Ndel1 palmitoylation was estimated to be 1.75 h (R2=0.985). Ndel1 is a very stable protein and the protein half-life was estimated to be 24 h (Figure 1H and Supplementary data). In primary cortical neurons the half-life of Ndel1 palmitoylation was estimated to be 2.3 h, as determined by the relative reduction of the signal after incubation of the cortical cultures with the palmitate analogue 2-bromopalmitate (2-Br), which inhibits palmitoylation (Webb et al, 2000; Figure 1I and J). Figure 1.Ndel1 and Nde1 are palmitoylated proteins. (A, B) Radioactive palmitate was incorporated in HEK293 cells co-transfected with (A) wild-type GFP–Ndel1 or (B) GFP–Nde1 and each of the palmitoylation enzymes. (C, D) In vivo palmitoylation of Ndel1 and Nde1 using the ABE (acyl-biotinyl exchange) method. Brain proteins from P7 mice (C, D) were extracted in a buffer containing N-ethyl malamide. (C) Ndel1 or (D) Nde1 were immunoprecipitated using anti-Ndel1 or anti-Nde1 antibodies. Palmitate was removed by addition of hydroxylamine (controls without hydroxylamine). BMCC-Biotin binding to the free thioester group enabled detection of the palmitoylated population of proteins by avidin–HRP. The positions of palmitoylated Ndel1 or Nde1 are indicated by small arrows. The western blots using anti-Ndel1, anti-DIC, or Nde1 antibodies indicated the position of Ndel1 (C, top), and similar amount of immunoprecipitated DIC (C, bottom) or Nde1 (D, bottom) proteins. (E, F) A band shift is noted when (E) PSD95 from brain lysate or (F) Ndel1 from CAD cell lysate are separated on SDS–PAGE after addition of neutral pH hydroxylamine (without HA−, with HA+,). Short arrows indicate the position of the nontreated bands, whereas the longer arrows indicate the positions of the hydroxylamine-treated bands. (G–K) Determination of Ndel1 palmitoylation half-life. (G, H) GFP–Ndel1 transfected in DHHC7 HEK293 inducible cell line, was metabolically labelled with 17-ODYA, followed by chase of palmitic acid. Ndel1 was immunoprecipitated and subjected to click chemistry, separated by SDS–PAGE, in-gel fluorescence was monitored (top panel), and the fluorescence was removed after HA treatment (middle panel). Similar amounts of Ndel1 were immunoprecipitated (lower panel). (H) Palmitoylation and protein half-life linear regression. The in-gel fluorescence intensity was normalized according to the amount of immunoprecipitated Ndel1 and plotted (data points are triangles and the linear regression is solid black line). Ndel1 protein half-life data (described in supplementary data) was ploted as well (data points are squares ±s.e.m. and the linear regression is a dashed line). Ndel1 is a very stable protein with an estimated half-life of about 24 h; Ndel1 palmitoylation half-life is about 1.7 h. (I, J) Half-life in cortical neurons. Cortical neurons were cultured in the presence of 2-bromopalmitate (2-Br) and palmitoylation at the indicated time points were determined by the ABE method. (J) The relative intensities after normalization with intensity of the immunoprecipitated protein were plotted against time and the half-life line is indicated. Download figure Download PowerPoint Next, the cysteine residue(s) undergoing S-palmitoylation were identified. Four Ndel1 cysteine residues were individually mutated (C203S, C273S, C293S, and C302S), and the mutated proteins were assayed for their capability to undergo palmitoylation (Figure 2A). The mouse Nde1 protein contains only one conserved cysteine residue, and it was mutated as well (C273S) (Figure 2B). The palmitoylation of both Ndel1 and Nde1 C273S mutant proteins was statistically significantly reduced using either DHHC2, DHHC3, or DHHC7, suggesting that this conserved site is the major site of palmitoylation on both proteins (Figure 2C and D). An alternative explanation will be that this site is required for palmitoylation of other sites. We conclude that both Ndel1 and Nde1 can be palmitoylated in vitro and in vivo by DHHC2, 3, and 7, and that C273 is important for palmitoylation. Figure 2.Identification of Ndel1 and Nde1 palmitoylated site. (A, B) Ndel1 (A) and Nde1 (B) are palmitoylated on cys 273 by DHHC2, 3, and 7. HEK293 cells co-transfected with wild-type GFP–Ndel1, or GFP–Nde1 or mutated on the indicated cysteines together with the indicated palmitoylation enzymes DHHC2, 3, and 7, were labelled with [3H]-palmitate. The cell lysates separated by SDS–PAGE were subjected to fluorography and expression levels of GFP–proteins were monitored by immunoblotting with an anti-GFP antibody. (C, D) The autoradiograms in (A, B) were subjected to quantification demonstrating that when Cys 273 is mutated, palmitoylation decreased. (E) Sequence comparison (BLAST) between Ndel1 and Nde1 of the amino acids surrounding Cys 273. Download figure Download PowerPoint Palmitoylation of Ndel1 affects its interaction with cytoplasmic dynein Palmitoylation may affect numerous processes, including protein–protein interactions. Our analysis depicted a single lipid modification that is not sufficient for membrane attachment (for review, see Resh, 2006). Indeed, Ndel1 palmitoylation did not grossly increase its relative proportion in the membrane as the relative proportion of wild type or C273S Ndel1 localization in the membrane or in the cytosol did not change after expression of the palmitoylation enzyme. Furthermore, the relative proportion of endogenous or transfected Ndel1 did not significantly change after expression of wild-type or mutated DHHC7 (Supplementary Figures S2 and S3). Nevertheless, we do not exclude the possibility of fine changes in the interaction of Ndel1 and some membranal subpopulations. Ndel1 is known to complex with LIS1, cytoplasmic dynein, and neurofilaments. The main palmitoylated cysteine in Ndel1 resides within the mapped interaction domain with cytoplasmic dynein in the C-terminal part of this protein (Sasaki et al, 2000), whereas the interaction with LIS1 has been localized to a different region within the coiled-coil domain (Derewenda et al, 2007; schematically presented in Figure 3A). Figure 3.The effect of Ndel1 or Nde1 palmitoylation on their interaction with dynein intermediate chain. (A) Schematic presentation of Ndel1 and its binding domains. The coiled-coil domain (amino acids 10–160, light grey) contains the mapped binding domain with LIS1 (amino acids 103–153, blue). The dynein-binding domain (amino acids 256–291, yellow) contains the palmitoylated site C273. (B) HEK293 cells were transiently transfected with GFP–Ndel1wt/C273S with or without HA–DHHC7. Ndel1 or DIC were immunoprecipitated. The interaction between Ndel1 and DIC is reduced when Ndel1 is palmitoylated (second lane from the left side). In the absence of the palmitoylation enzyme (DHHC7) or when Ndel1 cannot undergo palmitoylation (C273S), the interaction is increased. (C) Introduction of DHHC7 DN or Ndel1 C273S reduces Ndell palmitoylation as evident by 3H-palmitate incorporation. Similar amounts of the proteins were expressed evident by anti-GFP western blot. (D) Reduction in Ndel1 palmitoylaiton increases its interaction with DIC. More Ndel1 is co-immunoprecipitated with DIC after 2-Br treatment (upper row), similar amounts of DIC were immunoprecipitated and similar amount of Ndel1 were present in the lysates. Nevertheless, Ndel1 palmitoylation was decreased after 2-Br treatment evident by in gel fluorescence. The induction of DHHC7 in the HEK293 cell line was evident by western blot with the anti-HA tag. (E) The interaction between Nde1 and DIC is unaffected by palmitoylation. However, the interaction of mutant Nde1 (C273S) with DIC is diminished. The result is seen by both reciprocal immunoprecipitations: IP of DIC (top panel) and IP of Nde1 (bottom panel). Download figure Download PowerPoint We used immunoprecipitation to test whether Ndel1 palmitoylation affects its interaction with cytoplasmic dynein or with LIS1 in transfected cells. Palmitoylated Ndel1 (co-expressed with its palmitoylation enzyme DHHC7) exhibited reduced interaction with the cytoplasmic dynein complex (Figure 3B) when dynein was co-immunoprecipitated with Ndel1. In contrast, the unpalmitoylatable C273S-mutated version did not exhibit the observed reduction in interaction with the dynein complex, confirming that this effect is due to palmitoylation of Ndel1. The interaction between Ndel1 and LIS1 was not affected by palmitoylation, or by the C273 point mutation. Using metabolic labelling and click chemistry, we could demonstrate that Ndel1 palmitoylation is reduced in the presence of DHHC7-DN or when C273 is mutated (Figure 3C). Similar reduction in the interaction between Ndel1 and DIC was noted when Ndel1 was co-immunoprecipitated with DIC. In this case palmitoylation of Ndel1 was reduced in HEK293 cells expressing inducible DHHC7 after treatment with the well-characterized inhibitor of palmitoylation 2-Br (Jennings et al, 2009; Figure 3D). Unlike Ndel1, Nde1 co-immunoprecipitation with the dynein complex was not affected by palmitoylation (Figure 3E). Similar amounts of immunoprecipitated Nde1 in the presence or absence of the palmitoylation enzyme DHHC7 were observed. Nevertheless, cys 273 has an important role in mediating the binding of Nde1 to cytoplasmic dynein, as Nde1 C273S exhibited a 5.6-fold reduced interaction with DIC (paired Student's t-test, P=0.0004) regardless of the presence of the palmitoylation enzyme (Figure 3E). The difference between the behaviour of Nde1 and Ndel1 in that respect may be due to slight differences in the sequence of the amino acids surrounding C273 (Figure 2E). Amino acids in that region may also contribute to the interaction with dynein. Structural analysis of the complexes involving Nde1 or Ndel1 and dynein are likely to provide better insight into this issue. The interaction between Nde1 and LIS1 also was not affected by palmitoylation, and not by introduction of the C273S mutation (data not shown). Therefore, despite the high sequence similarity (72.6%) between Ndel1 and Nde1, our results uncovered a clear functional difference between these two paralog proteins. The idea that Nde1 and Ndel1 have non-overlapping functions in regard to palmitoylation-dependent dynein interactions, prompted us to carry out a phylogenetic analysis for Nde1 and Ndel1 from invertebrates and vertebrates (Supplementary Figure S4). The analysis revealed a clear distinction between the groups of Nde1 and Ndel1 proteins in vertebrates, whereas invertebrates (represented here by two different species of Drosophila) contain a single Nde1 protein. Interestingly, the possible divergence between the Protostomia (invertebrates) and the Deuterostomia (vertebrates) was estimated to occur 700–520 Mya (Erwin and Davidson, 2002) or between 580 and 520 Mya (Halanych, 2004). Therefore, it is likely that as sequences diverged, unique functionalities were adopted for the two proteins. Palmitoylation is a reversible modification; therefore, a plausible hypothesis is that the differential interaction of palmitoylated versus unpalmitoylated Ndel1 with cytoplasmic dynein is a new switch mechanism to modulate the activity of the molecular motor. This hypothesis was tested directly by monitoring the activity of cytoplasmic dynein using several cellular assays using different cells. The levels of Ndel1 and the palmitoylation enzymes vary in the different cells (Supplementary Figure S5). Ndel1 is highly expressed in the rat brain and in CAD cells; it is expressed at moderate levels in NIH3T3 cells and at very low levels in COS7 cells and HEK293 cells. DHHC7 expression is very low in NIH3T3 cells and HEK293 cells, and is better expressed in CAD, COS7, and brain lysate, whereas DHHC3 is expressed at high levels in NIH3T3 cells. Therefore, in some cases, we needed to transfect both Ndel1 and the palmitoylation enzyme to observe an effect. We demonstrated that the Ndel1 is palmitoylated when it is co-transfected with the palmitoylation enzyme and its palmitoylation is significantly reduced when Ndel1 (C273S) was co-transfected with the wild-type enzyme or when wild-type Ndel1 was co-transfected with the DN-enzyme in HEK 293 cells and COS7 cells (Figure 3C and Supplementary Figure S6). Cytoplasmic dynein and Golgi structure Several lines of evidence suggest that the perinuclear position of the Golgi is driven by cytoplasmic dynein-mediated transport. In cells lacking cytoplasmic dynein heavy chain (DHC), the Golgi is dispersed (Harada et al, 1998) and injection of anti-DHC2 antibody also led to dispersion of the Golgi complex (Vaisberg et al, 1996). In addition, depletion of cytoplasmic dynein prevented the centrosomal localization of exogenously applied Golgi-derived vesicles in partially permeabilized cells (Corthesy-Theulaz et al, 1992). Finally, overexpression of the dynamitin subunit of the dynactin complex, which dissociates the dynein complex, also led to the dispersion of the Golgi complex among other cellular phenotypes (Burkhardt et al, 1997). Similarly, the activity of Ndel1 and LIS1 in regulating dynein-mediated perinuclear clustering of the Golgi apparatus was demonstrated in Lis1- and Ndel1-null mouse embryonic fibroblasts (Sasaki et al, 2005). In tissue culture cells, this activity has been demonstrated using either Ndel1 mutants defective in LIS1 or DHC binding or by the silencing of Ndel1 using siRNA (Liang et al, 2004). The compactness of the juxtanuclear Golgi complex was measured in NIH3T3 cells by immunostaining with anti-Mannosidase II (an integral Golgi enzyme) antibodies (Figure 4). Palmitoylated Ndel1 significantly induced Golgi dispersion (Figure 4C–E) without any obvious effects on the microtubule cytoskeleton (data not shown). In addition, a portion of Ndel1 clearly localized to the Golgi (Figure 4A and C). In contrast, when endogenous palmitoylation is reduced by expression of DHHC7-DN, the size of the Golgi is not changed (Figure 4A, B and E). Similarly, palmitoylated Nde1 or expression of individual components Ndel1, Nde1, or the palmitoylation enzyme DHHC7 alone had no effect on Golgi distribution (Figure 4E). These results are consistent with our previous findings, suggesting that strong interaction of unpalmitoylated Ndel1, but not Nde1, with cytoplasmic dynein is required for the perinuclear positioning of the Golgi. Figure 4.Palmitoylated Ndel1 reduces cytoplasmic dynein activity resulting in dispersed Golgi. NIH3T3 cells were transfected with all combinations of GFP–Ndel1 (wild type or C273S) with DHHC7 WT or C160S mutant (DHHC7-DN), or combinations of GFP–Nde1(wild type or C273S) and/or DHHC7 (wild type or DN). (A, B) When Ndel1 palmitoylation is low in the presence of DHHC7-DN, (B) the Golgi immunostained with anti-mannosidase II antibodies, is compact (A, merged picture). (C, D) When Ndel1 is palmitoylated (D), in the presence of DHHC7, the Golgi is more distributed (D, merge C). (E) The Golgi area in the indicated transfected cells was measured and subjected to statistical analysis. Co-transfection of Ndel1 and the palmitoylation enzyme DHHC7 increased the area of the Golgi (ANOVA, P<0.0001, n=21). Error bars indicate s.e.m. value. Size bar is 15 μm. Download figure Dow
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