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Vol. 20, Issue 21, 4586-4595, November 1, 2009
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Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802
Submitted October 23, 2008;
Revised September 4, 2009;
Accepted September 10, 2009
Monitoring Editor: David G. Drubin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Crisp et al., 2006). One partner is an outer nuclear membrane (ONM) protein that binds to a component of the actin or microtubule cytoskeleton and the second is an inner nuclear membrane (INM) protein that binds to the luminal domain of the ONM protein as well as an immobile nuclear structure, like the lamina. These ONM–INM membrane protein pairs can thus connect structural components of the nucleus and cytoplasm across the double membrane of the nuclear envelope, and they have a critical and evolutionarily conserved role in moving or anchoring nuclei within the cytoplasm (Starr, 2007).
In each case where a pair of proposed INM and ONM partner proteins has been identified, the ONM candidate contains a Klarsicht/ANC-1/Syne/homology (KASH) domain, which consists of transmembrane domain and 
30 amino acids that follow it at the C terminus of the protein (Starr and Han, 2002; Starr and Fischer, 2005). Similarly, the INM protein partners contain a conserved domain; the Sad-1/UNC-84 (SUN) domain is localized at the C terminus of the INM partner. The model for topology of these proteins suggests that the KASH domain resides in the space between the INM and ONM where it mediates the interaction with the INM partner. The SUN domain is also proposed to lie in the intermembrane space. According to the model, the N termini of SUN domain proteins lie in the nucleus and are important for attachment of the complex to the lamina or chromatin. Whereas several ONM–INM protein pairs are proposed to fit this model for bridging the nuclear envelope and various aspects of this model have been tested for some of the KASH–SUN partners (Padmakumar et al., 2005; Crisp et al., 2006; Haque et al., 2006; McGee et al., 2006), the localization, topology, and binding interactions of a SUN–KASH pair have rarely been thoroughly and simultaneously examined.
To investigate the SUN-KASH model for bridging the nuclear envelope, we have focused on the SUN-1 and ZYG-12 pair. We previously reported that zyg-12 mediates the essential attachment of the centrosome to the nucleus in early Caenorhabditis elegans embryos (Malone et al., 2003). zyg-12 mutants also showed nuclear positioning, migration, and chromosome segregation defects. Based on the observation that ZYG-12 localizes to the nuclear envelope and interacts with cytoplasmic dynein, it is proposed to localize to the ONM, with access to the cytoplasm (Malone et al., 2003). ZYG-12 is also observed on the centrosome, and ZYG-12 homo-dimerizes. Thus, we hypothesize that ZYG-12 mediates the association between the nucleus and the centrosomes through homotypic interaction. Given that the outer nuclear envelope is thought to be contiguous with the endoplasmic reticulum, ONM-specific localization of ZYG-12 is critical for its function. However, the molecular mechanism which restricts ZYG-12 to the ONM has not been fully elucidated.
ZYG-12 nuclear envelope localization requires the SUN domain protein, SUN-1 (Malone et al., 2003). Because SUN-1 interacts with nuclear lamins, it is likely to localize to the INM (Fridkin et al., 2004). We propose that the ZYG-12/SUN-1 pair is localized to the nuclear envelope and functions to mediate centrosome-nucleus attachment. Although there is evidence that ZYG-12/SUN-1 are a bona fide KASH/SUN pair, direct evidence of their interaction and their topologies is missing. Thorough investigation of the localization, topology, and interactions of the two proteins will provide important insight into the model for function of SUN–KASH pairs as well as the molecular mechanism for linkage of the centrosome and the nucleus. ZYG-12 is also an interesting model for SUN–KASH pair investigations because the KASH domain of ZYG-12 is significantly shorter than other identified family members and contains only four of the eight absolutely conserved residues in other KASH domains (Figure 1A). Thus, our experiments also lend important insight into the potential diversity of KASH domain sequences.
We took advantage of in vivo nuclei that express endogenous ZYG-12 and SUN-1 in combination with in vitro assays to determine that ZYG-12 resides in the outer membrane of the nuclear envelope in vivo and directly interacts with inner nuclear membrane protein SUN-1 using a fully functional, although divergent, mini KASH domain. We further show that ZYG-12 has restricted mobility at the nuclear membrane by using fluorescence recovery after photobleaching (FRAP) analysis and that SUN-1 is sufficient for ZYG-12 localization via ectopic expression of both proteins in mammalian cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) except qaIs3502[unc-119(+) + pie-1::YFP::LMN-1 + pie-1::CFP::H2B], which was grown at 25°C. We used the pie-1 promoter and enhancer system (vector pFJ1) to express green fluorescent protein (GFP) reporter proteins in the germ line and early embryos (Strome et al., 2001). See Supplemental Table S1 for vector construction. We performed transformations by using the microparticle bombardment system (Praitis et al., 2001, Strome et al., 2001). We performed RNA interference (RNAi) by feeding (see Supplemental Data).
Proteinase K Protection Assay
We cloned full-length sun-1cDNA into pCITE2b (Novagen, Madison, WI) (details upon request) and used linearized vector for in vitro transcription using Megascript 7 (Ambion, Austin, TX). We synthesized SUN-1 using Flexi rabbit reticulocyte lysate (Promega, Madison, WI) in the absence or presence of canine microsomal membranes (Promega). Five microliters of translated protein was digested with 4 µg/ml proteinase K on ice for 30 min followed by sequential addition of 1 mM phenylmethylsulfonyl fluoride (PMSF) and 30 µl of 1x SDS-polyacrylamide gel electrophoresis sample buffer containing dithiothreitol. The protected fragments were analyzed by Western blotting.
Epitope Accessibility Assay (EAA)
Gonads of wild-type animals were cut open on coverslips and immobilized on poly-L-lysine–coated slides. We fixed gonads with 4% formaldehyde in phosphate-buffered saline (PBS) and immunostained using 
-SUN-1 antibody, which was produced against bacterially expressed protein containing the C-terminal 242 amino acids of SUN-1 (generous gift from Abby Dernburg, University of California–Berkeley) or -ZYG-12 rat polyclonal antibodies (Malone et al., 2003) in the absence or presence of 1% Triton X-100. Samples were blocked with 0.5% bovine serum albumin, incubated with primary antibodies at 4°C overnight, washed with cold PBS, and incubated with secondary antibodies (Alexa Fluor 488 and 568; Invitrogen, Carlsbad, CA) for 1 h at room temperature. We acquired images using ECLISPE 90i wide-field microscopy (Nikon, Melville, NY) and SimplePCI software (Compix, Irvine, CA) and processed images using PhotoShop software (Adobe Systems, Mountain View, CA).
Fluorescence Protease Protection (FPP) Assay
Gonads from qaIs3502 and wild-type hermaphrodites were cut, immobilized on poly-L-lysine–coated coverslips and fixed with 4% formaldehyde. They were washed with cold PBS and incubated with 1 mg/ml trypsin in PBS for 10 min at 4°C. Samples were then washed with cold PBS containing 1 mM PMSF and 1 µg/ml aprotinin and transferred to poly-L-lysine–coated slides. We immunostained in the presence of Triton X-100 by using 3E6 monoclonal antibodies against GFP (Invitrogen), -SUN-1, and -ZYG-12 antibodies as described above.
Yeast Two-Hybrid Assay
We used a split-ubiquitin based yeast two-hybrid system (Fetchko and Stagljar, 2004). See Supplemental Data for details.
Fluorescence Recovery after Photobleaching
We used GFP fusions to endoplasmic reticulum (ER) resident protein signal peptidase SP-12 (Rolls et al., 2002; Poteryaev et al., 2005), to all isoforms of ZYG-12 (Malone et al., 2003), and to SUN-1 (see above) for FRAP analysis. We examined nuclei of developing oocytes in the region of the gonad just proximal to the turn with a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Thornwood, NY) with the Plan-Apocromat 63x 1.4 oil differential interference contrast (DIC) objective (Carl Zeiss MicroImaging). We excited with the 488-nm line of an Ar laser and detected emitted light with a 505-nm long pass filter. We placed extruded gonads on a 2% agarose pad and bleached a rectangular portion of the nucleus by using a 488-nm laser line at 100% power. The nuclei were monitored at 10-s intervals up to 300 s. We measured the intensity of total and bleached regions by using NIH ImageJ (National Institutes of Health, Bethesda, MD) and normalized as described earlier to account for the bleaching during monitoring (Ostlund et al., 2006) using the formula Irel = T0It/TtI0, where T0 is total fluorescence of the image before bleaching, I0 is fluorescence from the bleached region before bleaching, Tt is total fluorescence of the image at time point t, and It, is fluorescence from the bleached region at time point t. At least six independent bleaching experiments were combined for each GFP reporter, and the normalized values are means ± SD.
HeLa Cell Culture and Transfection
HeLa cells were maintained in DMEM/Ham's F-12 with 10% fetal bovine serum (FBS) (Invitrogen), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Mediatech, Herndon, VA). pECFP-ER (Clontech, Mountain View, CA) was used as the ER marker. Full-length sun-1 and zyg-12 B and C cDNAs were cloned into pEYFP C1 and pECFP C1, respectively (Supplemental Table S3). Plasmid DNAs were transfected into HeLa cells by using Effectene Transfection Reagent (QIAGEN, Valencia, CA). One microgram of DNA in 150 µl of EC buffer was mixed with 8 µl of Enhancer and incubated for 5 min at room temperature (RT). After adding 25 µl of Effectene Transfection Reagent and incubating for 10 min at RT, DMEM/Ham's F-12 with 10% FBS was added to the mixture. Subsequently, the mixture was transferred to 5 x 105 HeLa cells seeded onto a six-well culture plate 1 d prior and incubated at 37°C for 4 h. Cells were washed with PBS and incubated for 2 d in DMEM/Ham's F-12 with 10% FBS. Cells were observed using Axiovert 200M microscope (Carl Zeiss MicroImaging) with Chroma 41028 filter for yellow fluorescent protein (YFP) and Chroma 31044 V2 filter for cyan fluorescent protein (CFP) (Chroma Technology, Brattleboro, VT). The image stacks of the Z-axis were taken and deconvolved using AxioVision software (Carl Zeiss MicroImaging) and processed using PhotoShop CS software (Adobe Systems).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KASH domains results in the loss of nuclear membrane specific localization (Padmakumar et al., 2005; Ketema et al., 2007). C. elegans ZYG-12 has three isoforms (A, B, and C) (Malone et al., 2003). Two of the three ZYG-12 isoforms (B and C) contain transmembrane domains and are proposed to localize to the ONM (Malone et al., 2003) but do not contain all the hallmarks of known KASH domains. The third isoform, A, has a similar N terminus, but it has no transmembrane domain and is localized exclusively to the centrosome. Like ONM-localized KASH domain proteins, ZYG-12 B and C have a predicted transmembrane domain near the C terminus, followed by a short tail. However, the tail contains only four residues that are found in other KASH domains (Figure 1A). In addition, the mini KASH domain contains only two prolines of the -PPPT motif that is required for the nuclear envelope localization of human Syne-2. To test whether the divergent mini KASH domain of ZYG-12 B and C is functional and sufficient to localize the protein to the nuclear envelope, we expressed GFP-tagged truncations of ZYG-12 containing the mini KASH domain in embryos (Figure 1B). ZYG-12 B 
2 and ZYG-12 C 2 containing the mini KASH domains of ZYG-12 B and C, respectively, localized to the plasma membrane. However, ZYG-12 B 1 and ZYG-12 C 1 containing the mini KASH and 52 amino acids of predicted coiled-coil domain of ZYG-12 B and C, respectively, localized to the nuclear envelope and plasma membrane (Figure 1C). This result indicates that the mini KASH domain together with a partial coiled-coil domain is sufficient for the nuclear envelope localization of ZYG-12 B and C. Because the coiled-coil domain of ZYG-12 is responsible for self-dimerization (Malone et al., 2003), we tested the possibility that ZYG-12 B 1 and ZYG-12 C 1 might interact with endogenous ZYG-12 at the nuclear envelope to achieve nuclear envelope localization. We introduced double-stranded (ds)RNA that specifically targets the N-terminal sequence of ZYG-12 to reduce endogenous ZYG-12 without affecting the ZYG-12::GFP truncations (Figure 1B). Depletion of endogenous ZYG-12 was successful as shown by the detached centrosome phenotype (Figure 1 C, asterisk). Nonetheless, nuclear envelope localization of ZYG-12 B 1 and ZYG-12 C 1 was maintained when endogenous ZYG-12 was depleted. None of the truncations tested localized to the centrosome in contrast to the full-length ZYG-12 B and C that localize to both the nuclear envelope and the centrosome (Malone et al., 2003). This indicates that the N terminus of the ZYG-12 is required for centrosome localization.
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1 and ZYG-12 C 1 to the nuclear membrane requires SUN-1. We depleted SUN-1 via RNAi and found that nuclear envelope localization of ZYG-12 B 1 and ZYG-12 C 1 was completely lost, whereas plasma membrane localization was still intact. These results indicate that ZYG-12 truncations are targeted to the nuclear envelope by SUN-1 and that plasma membrane localization is probably nonspecific. We conclude that addition of the coiled-coil domain to the mini KASH domain is sufficient to localize ZYG-12 to the nuclear envelope and that this localization is dependent on SUN-1. These results suggest that the divergent mini KASH domain of ZYG-12 B and C functions like other KASH domains.
ZYG-12 and SUN-1 Are Type II Integral Membrane Proteins That Localize to the Outer and Inner Membrane of the Nuclear Envelope, Respectively
We predict that the ZYG-12 C-terminal mini KASH domain interacts with the C terminus of SUN-1 in the lumen between the INM and ONM. To test whether the proteins are type II integral membrane proteins that are inserted in the membrane with their C termini in the lumen, we performed three independent assays.
First, we examined the membrane orientations of ZYG-12 and SUN-1 at the outer and the inner membranes of the nuclear envelopes, by using an FPP assay (Lorenz et al., 2006). Trypsin is a small protease that can freely diffuse into nuclei through the nuclear pore complex resulting in digestion of proteins on the outside and the inside of the nuclear envelope. Regions of proteins in the lumen between the ONM and INM are protected from trypsin cleavage. Thus, analysis of protein fragments protected from trypsin cleavage reveals information about the topology of proteins. We extruded gonads from wild-type (Figure 2C) animals and animals expressing YFP::LMN-1 (Figure 2B) and used intrinsic fluorescence to detect YFP::LMN-1 and antibodies directed against C-terminal epitopes to detect SUN-1 (Figure 2A). Without trypsin treatment, both lamin::YFP and SUN-1 were detectable (Figure 2B, first column). Trypsin treatment reduced Lamin::YFP to undetectable levels, as expected. However, the C-terminal fragment of SUN-1 was protected from protease cleavage (Figure 2B, second column), indicating that the C terminus of SUN-1 resides in the lumen of the nuclear envelope. In similar experiments using the SUN-1 antibody and the ZYG-12 antibody (Figure 2A), we confirmed that the N terminus of ZYG-12 is accessible to trypsin (Figure 2C). Thus, the C termini of both ZYG-12 and SUN-1 reside in the nuclear envelope lumen.
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-ZYG-12 N terminus and -SUN-1 C terminus antibodies (Figure 2A) in the absence and the presence of Triton X-100. In the presence of Triton X-100, the ZYG-12 and SUN-1 epitopes were accessible to antibody as expected, resulting in clear detection of signal at the nuclear envelope (Figure 3, A, C, and E). In contrast, in the absence of the detergent, only the ZYG-12 epitope was accessible to the antibody (Figure 3, B, D, and F), indicating that the N terminus of ZYG-12 is on the outside of the nucleus. The lack of SUN-1 signal in the absence of Triton X-100 is consistent with a luminal localization of its C terminus.
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) and hidden Markov model for topology prediction (HMMTOP) (Tusnady and Simon, 1998, 2001) predict only an N-terminal transmembrane domain (TM) domain for SUN-1, whereas TMpred (Hoffman et al., 1993) predicts a second C-terminal TM domain. We synthesized full-length SUN-1 in vitro in the absence or presence of canine pancreatic microsomal membranes and treated with proteinase K in the absence or presence of Triton X-100. The protected fragments of SUN-1 were then visualized by Western blot analysis using -SUN-1 C terminus (Figure 2A). The size of the protected fragment of SUN-1 was 40 kDa (Figure 4D, arrow), which is consistent with the size of the portion of SUN-1 to the C terminus of the first predicted TM (Figure 4A). The exclusive use of TM2 would have produced a 23-kDa fragment, and use of both TMs would produce 13- and 25-kDa fragments (Figure 4C). The protected fragment was not detectable from the reactions without microsomes or with Triton X-100, indicating that microsomes prevent digestion. This result indicates that the first TM domain of SUN-1 is used, and SUN-1 is a type II integral membrane protein with the C terminus in the lumen.
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), and we showed that the C termini of ZYG-12 and SUN-1 are localized to the lumen between the ONM and INM. Thus, an interaction between ZYG-12 and SUN-1 in the lumen is a possible mechanism for ONM-specific localization of ZYG-12. We used a yeast two-hybrid assay to test whether SUN-1 physically interacts with ZYG-12. Because SUN-1 and ZYG-12 are integral membrane proteins, which potentially interact in the luminal space of the nuclear envelope, we took advantage of the DUALmembrane yeast two-hybrid system based on split-ubiquitin (Stagljar et al., 1998; Fetchko and Stagljar, 2004).
We expressed full-length ZYG-12 B and C and SUN-1 and various SUN-1 truncations and mutants as bait constructs (Figure 5A). We expressed C-terminal portions of ZYG-12 B (86 amino acids) and C (70 amino acids) and SUN-1 as prey. We showed previously that full-length ZYG-12 A binds to itself by using the internal coiled-coil domain (Malone et al., 2003). Full-length ZYG-12 B and C contain coiled-coil domains identical to ZYG-12 A and bind to each other (data not shown) but not to ZYG-12 B, C1 that lack N terminus (Figure 5A). SUN-1 fragments (SUN-1 3, 4, and 5) that lack C-terminal region including the conserved SUN domain were able to interact with ZYG-12 B, C1 (Figure 5A). Interestingly, SUN-1 fragments that had only 40 more amino acid residues after the first transmembrane domain (SUN-15) were able to interact with ZYG-12 truncations. To further define the region of SUN-1 that interacts with ZYG-12, we created SUN-1 bait fragment that has only five additional amino acid resides after first transmembrane domain (SUN-16) and a bait with an internal deletion that retains the five amino acids and the conserved SUN domain (SUN-17). Neither of these was able to interact with ZYG-12 truncations. Other internal deletions (SUN-1 8, 9, and 10) that maintain 40 amino acids and SUN domain interacted with ZYG-12. These results indicate that the interaction with ZYG 12 requires the internal domain of SUN-1 (residues 123325) and does not require the conserved SUN domain. In addition, to test whether residues 123–163 are necessary for the interaction with ZYG-12 truncation, we made SUN-1 fragment (SUN-111) that lacks this region. This fragment was still able to interact with ZYG-12 truncations, indicating that the 40-amino acid stretch after the first transmembrane domain is not the sole requirement for the interaction. One explanation of the data is that there are two regions within the internal domain that can each independently interact with ZYG-12, one region comprises amino acids 123–163, and the other is between amino acids 163 and 325. The data do not support any interaction between the SUN domain and ZYG-12.
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2, which lacks the N-terminal 158 amino acids, including the first predicted transmembrane domain, self-activated, as expected from a bait protein that cannot target to the ER membrane and can thus enter the nucleus and activate the target genes. In contrast, SUN-11, which preserves the first putative transmembrane domain, did not self-activate. This finding is consistent with our proteinase K protection assay, indicating the first predicted transmembrane domain of SUN-1 is the only functional one. We conclude that SUN-1 directly interacts with ZYG-12 and the interaction occurs between the internal domain of SUN-1 and the mini KASH domain of ZYG-12. Because SUN-1 has two putative coiled-coil domains, we examined whether SUN-1 can bind to itself. SUN-1 constructs that contained the coiled-coil domains interacted with full-length SUN-1 in the prey, whereas SUN-1 truncations that lack this region did not (Figure 5B). This result suggests that SUN-1 binds to itself using the internal coiled-coil domains.
ZYG-12 Is Immobile at the Outer Membrane of the Nuclear Envelope
We have shown that ZYG-12 localizes to the outer membrane of the nuclear envelope in a SUN-1–dependent manner. In addition, ZYG-12 does not diffuse throughout the ER even though the outer membrane of the nuclear envelope is contiguous with the ER. We hypothesized that if ZYG-12 were restricted to the outer membrane of the nuclear envelope by an interaction with the inner nuclear membrane protein SUN-1, ZYG-12 would behave like an inner membrane protein in mobility tests. To test this prediction, we performed FRAP analysis (Figure 6). As expected, the ER resident transmembrane protein signal peptidase (SP-12) quickly recovered within 30 s after bleaching. In contrast, the inner nuclear membrane protein SUN-1 did not recover after bleaching, indicating that its movement in the membrane is restricted like other inner membrane proteins. Similarly, we found that ZYG-12 at the outer nuclear membrane was not able to recover after bleaching. These results indicate that ZYG-12 mobility in the outer nuclear membrane is restricted and support our model that ZYG-12 is specifically localized to the outer membrane of the nuclear envelope through the interaction with inner nuclear membrane protein SUN-1.
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). To determine whether interaction with SUN-1 is sufficient to localize ZYG-12, we tested whether SUN-1 could recruit ZYG-12 in a heterologous system. We independently expressed ZYG-12 and SUN-1 in human HeLa cells (Figure 7, B–D) and then coexpressed the proteins (Figure 7, E–J). When full-length ZYG-12 B or ZYG-12 C is expressed in HeLa cells alone, it localizes diffusely to ER and the nuclear envelope (Figure 7, C and D). This localization pattern is very similar to that of an ER marker protein (Figure 7A). Unlike human Sun1, which is evenly distributed around the nuclear membrane when ectopically expressed in HeLa cells (Padmakumar et al., 2005), full-length CeSUN-1 tagged with YFP at its N terminus is clustered in patches near the HeLa nuclear membrane (Figure 7B). When SUN-1 is coexpressed with ZYG-12, ZYG-12 B and C are recruited to juxtanuclear patches where CeSUN-1 is localized, and the ER localization of ZYG-12 B and C was dramatically reduced to an almost undetectable level (Figure 7, E–J). The CeSUN-1 localization around the nuclei was unchanged during coexpression with ZYG-12. These results indicate that SUN-1 is sufficient to colocalize ZYG-12 and restrict its subcellular localization when they are coexpressed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1) were sufficient for the localization to the nuclear envelope, and SUN-1 was required for this localization. Although we subsequently found that the luminal domain of ZYG-12 binds directly to SUN-1, this region of the protein was not sufficient to localize a short fusion of the transmembrane domain with GFP to the NE. It is possible that short fusions of the C terminus of ZYG-12 to GFP do not insert correctly into membranes, or ZYG-12 may need part of the coiled coil to dimerize in order to localize to the NE.
Topology of ZYG-12 and SUN-1
The model (Figure 8) of KASH-SUN protein pairs coupling nucleoskeleton and cytoskeleton predicts that these localize to the outer and inner membrane of the nuclear envelope, respectively, with their C termini in the luminal space of the nuclear envelope where they bind one another. We used three assays to rigorously test whether ZYG-12 and SUN-1 adopt the topology and localization that fits this model. The most advantageous aspect of our approach was that we used nuclei from C. elegans gonads that express endogenous ZYG-12 and SUN-1 for two of the assays. Because the gonad is syncytial, we could avoid treating cells with digitonin to selectively permeabilize plasma membrane and could instead cut open the gonad to expose the nuclear surface. The EAA and FPP assays clearly show that endogenous ZYG-12 and SUN-1 are type II integral membrane proteins that localize to the outer and inner membrane of the nuclear envelope, respectively. There are several observations that support these findings. First, ZYG-12 has single transmembrane domain and is required for cytoplasmic motor protein Dynein nuclear envelope localization (Malone et al., 2003). Second, ZYG-12 also interacts with Dynein light intermediate chain. Third, SUN-1 binds to the nuclear lamina (Fridkin et al., 2004). Fridkin et al. (2004) reported that SUN-1/Matefin has two predicted transmembrane domains and localizes to the inner nuclear membrane with its N- and C-terminal ends inside of the nucleus. Our data, however, clearly suggest that SUN-1 has a single functional transmembrane domain with its C-terminal part, including the SUN domain, in the lumen of the nuclear envelope.
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). Previous studies have provided conflicting evidence on whether the SUN domain is responsible for interaction with the KASH partner. One study showed that SUN domain of human Sun2 is required for the interaction with human Nesprin-2 KASH domain (Stewart-Hutchinson et al., 2008). Our results are more consistent with another study that showed mammalian Sun1 utilizes internal luminal domain without SUN domain to interact with the KASH domain of Nesprin-1 (Padmakumar et al., 2005). Recently, novel C. elegans KASH protein KDP-1 was identified (McGee et al., 2009). KDP-1 localizes to the nuclear envelope in the germline in a SUN-1–dependent manner and interacts with SUN-1. It would be very interesting to examine whether SUN-1 uses different domains to interact with different KASH binding partners.
Roles of ZYG-12 (KASH) and SUN-1 (SUN) Pair in Centrosome–Nucleus Attachment
The identification of ZYG-12 as a protein with a fully functional, albeit divergent, KASH domain that lacks previously noted conserved residues means that other KASH domain proteins may have been missed in homology searches. Furthermore, ZYG-12 is the only protein to be identified that has both Hook and KASH domains. Hook proteins are thought to link microtubules and organelles and generally have N-terminal cytoskeleton binding Hook domains, internal coiled-coil domains for homodimerization, and C-terminal cytoplasmic organelle binding domains (Kramer and Phistry, 1999; Sunio et al., 1999). The localization of ZYG-12 to the ONM via its KASH domain and to the centrosome via its N terminus is an interesting twist on organelle–cytoskeleton linkage by Hook proteins—the dimerization of these two populations of ZYG-12 is responsible for centrosome anchoring to the NE in C. elegans embryos (Malone et al., 2003). The centrosomal binding partner of ZYG-12 has not yet been identified.
Although there are no obvious homologues of ZYG-12 in other organisms, human hook2 was found to reside at the centrosome by interacting with the centrosomal protein Centriolin/CEP110 by using its C-terminal domain (Szebenyi et al., 2007). Disruption of hook2 resulted in abnormal microtubule organization. Interestingly, the N-terminal region of hook2 is most similar to the N-terminal dynein-binding region of ZYG-12, suggesting a close relationship between these two proteins. However, hook2 does not localize to the ONM and is not used for anchoring the centrosome to the nucleus.
This means that the mechanism of nucleus-centrosome interaction is still unknown in many cells. In mammals, the INM protein emerin seems to be important as centrosomes in emerin-null or -deficient human dermal fibroblasts from X-linked Emery Dreifuss muscular dystrophy patients were detached from the nucleus (Salpingidou et al., 2007). In addition, emerin was found to be localized to the outer membrane of the nuclear envelope and interact with tubulin. How it would hold on to the centrosome has not been determined. These findings suggest that C. elegans and humans may use the same strategy for centrosomal attachment to the nucleus that involves a type II NE protein that can bind to microtubules or microtubule motors.
Using information about centrosome–nucleus interactions it should be possible to identify candidate proteins that mediate this link in other cells and organisms. Any NE protein that binds microtubules or any centrosomal protein that has a transmembrane domain at its C terminus could play a role in this process.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: IL Minn, (iminn1{at}jhmi.edu).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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