ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4904–4917 doi:10.1242/jcs.155408 RESEARCH ARTICLE A newly identified myomegalin isoform functions in Golgi microtubule organization and ER–Golgi transport Zhe Wang, Chao Zhang and Robert Z. Qi* ABSTRACT the formation of ER–Golgi cargo carriers that associate with dynein–dynactin to move along microtubules towards the Golgi The Golgi of mammalian cells is known to be a major microtubule- (Presley et al., 1997; Scales et al., 1997; Watson et al., 2005). organizing site that requires microtubules for its organization and The Golgi serves as a major microtubule-organizing center protein trafficking. However, the mechanisms underlying the (Chabin-Brion et al., 2001; Efimov et al., 2007; Miller et al., 2009; microtubule organization of the Golgi remain obscure. We used Rivero et al., 2009). For example, almost half of all cellular immunoprecipitation coupled with mass spectrometry to identify a microtubules originate from the Golgi in human retinal pigment widely expressed isoform of the poorly characterized muscle protein epithelial RPE1 cells (Efimov et al., 2007). Moreover, microtubule myomegalin. This newly identified isoform, myomegalin variant 8 nucleation at the Golgi does not require centrosomes, and it (MMG8), localized predominantly to cis-Golgi networks by interacting depends instead on c-tubulin (Efimov et al., 2007), the principal with AKAP450 (also known as AKAP9), and this interaction with microtubule nucleator in cells, which exists in the form of c-tubulin AKAP450 was required for the stability of both proteins. Disrupting complexes (cTuCs). The cis-Golgi proteins AKAP450 (also known MMG8 expression affected endoplasmic reticulum (ER)-to-Golgi as AKAP9, AKAP350, CG-NAP and hyperion) and GMAP210 trafficking and caused Golgi fragmentation. Furthermore, MMG8 (also known as TRIP11) have been proposed to be involved in c- associated with c-tubulin complexes and with the microtubule plus- tubulin recruitment to the Golgi and thus in the Golgi-associated end tracking protein EB1 (also known as MAPRE1), and was nucleation of microtubules (Rı´os et al., 2004; Rivero et al., 2009; required for the Golgi localization of these two molecules. On the Vinogradova et al., 2012). Microtubules originating from the Golgi Golgi, c-tubulin complexes mediated microtubule nucleation, are required for Golgi ribbon assembly, directional trafficking and whereas EB1 functioned in ER-to-Golgi trafficking. These results cell motility (Miller et al., 2009; Rivero et al., 2009). indicate that MMG8 participates in Golgi microtubule organization The growing tips of microtubules accumulate a diverse group and thereby plays a crucial role in the organization and function of the of proteins called plus-end tracking proteins (+TIPs) (Akhmanova Golgi. and Steinmetz, 2008). To track microtubule plus-ends, almost all +TIPs must interact with the end-binding (EB) proteins, among KEY WORDS: Golgi, Microtubule, Myomegalin, Protein trafficking which EB1 and EB3 (also known as MAPRE1 and MAPRE3, respectively) display similar tip-tracking properties, whereas EB2 (also known as MAPRE2) appears to be distinct from the other INTRODUCTION two proteins and, relative to them, exhibits considerably weaker The Golgi is a membranous organelle that plays a pivotal role in tip-tracking activity (Komarova et al., 2009). In one class of protein post-translational modification, sorting and transport. The +TIPs, an SxIP motif surrounded by basic and serine-rich assembly, positioning and function of the Golgi require an intact sequences is present that is required for the interaction of these microtubule cytoskeleton (Lippincott-Schwartz, 1998; Rios and +TIPs with the end-binding homology (EBH) domain of EB1 Bornens, 2003; Su¨tterlin and Colanzi, 2010). In interphase animal (Honnappa et al., 2009). EB1, the prototypic member of the EB cells, the Golgi exhibits a crescent-moon-shaped ribbon-like family, is detected at all growing microtubule tips, and the +TIPs morphology in the perinuclear region that typically surrounds the that are localized in association with EB1 at the microtubule plus- centrosome. When cells divide, the Golgi undergoes fragmentation ends perform diverse functions, including regulating microtubule and then reassembles during the late stages of mitosis. During dynamics and microtubule attachment to subcellular targets reassembly, microtubules derived from the Golgi and centrosomes (Akhmanova and Steinmetz, 2008). Although microtubules enable the Golgi to form a continuous ribbon structure featuring associate with the Golgi, whether EB1 and other EB proteins Golgi ministacks positioned near the center of cells (Miller et al., localize and function at the Golgi remains unknown. 2009). In the secretory pathway used by cells, proteins are In a yeast two-hybrid screen, myomegalin (myomegalin isoform transported from the endoplasmic reticulum (ER) to the Golgi, 1, MMG1; GenBank accession NP_055459) was cloned as a where they are eventually sorted into post-Golgi carriers. The ER- protein that interacted with cyclic nucleotide phosphodiesterase 4D to-Golgi transport of proteins is initiated by the packaging of cargo and thus was referred to as a phosphodiesterase 4D-interacting into COPII-coated vesicles at ER-exit sites, and it is followed by protein (Verde et al., 2001). The mmg1 sequence encodes a ,230- kDa protein that is expressed in heart and skeletal muscles. In Division of Life Science and State Key Laboratory of Molecular Neuroscience, The GenBank databases, MMG1 is the only protein present that is a Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, homolog of CDK5RAP2, a human microcephaly-related protein Hong Kong, China. that is involved in microtubule organization on centrosomes and in *Author for correspondence ([email protected]) microtubule regulation at growing microtubule tips (Choi et al., 2010; Fong et al., 2008; Fong et al., 2009). Here, we report that a Received 17 April 2014; Accepted 1 September 2014 newly identified nonmuscle MMG isoform, MMG8, functions in Journal of Cell Science 4904 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 4904–4917 doi:10.1242/jcs.155408 Golgi microtubule organization and ER-to-Golgi trafficking. Our products. However, MMG1 was not detected in any of these cell results reveal that MMG8 is a widely expressed protein that cultures (supplementary material Fig. S1D), although it was targets to the cis side of the Golgi by interacting with AKAP450. recognized by the 443M antibody in rat heart tissue extracts. On the Golgi, MMG8 is involved in recruiting cTuCs to promote To determine the subcellular localization of MMG8, we microtubule nucleation and also in tethering EB1 to enable immunostained HeLa cells with the MMG8-specific antibody. microtubule-tip capture and efficient ER-to-Golgi trafficking. MMG8 staining was highly enriched in the Golgi, and a weak Therefore, MMG8 plays a key role in Golgi microtubule general staining was detected in the cytoplasm (Fig. 1D). organization, which is required for efficient ER-to-Golgi Moreover, the distribution of the MMG8 signal on the Golgi trafficking and Golgi organization. was similar to that of the cis-Golgi protein GM130 (also known as GOLGA2), but it did not merge with the staining of the trans- RESULTS Golgi protein TGN46 (also known as TGOLN2) (Fig. 1D), Identification of MMG8 suggesting that MMG8 resides on the cis side of the Golgi. To detect MMG proteins in proliferating cell cultures, RT-PCR During cytokinesis, MMG8 appeared in both Golgi twins was performed using oligonucleotide primers targeting the human (Fig. 1D). mmg1 sequence encoding amino acids 474–762, a region present in other large MMG variants found in gene databases. This MMG MMG8 functions in Golgi organization and ER-to- sequence was specifically amplified from the total RNA extracted Golgi trafficking from HeLa cells (supplementary material Fig. S1A), and the To investigate the function of MMG8, we used RNA amplified product was verified through sequencing. To generate interference (RNAi) to suppress MMG8 expression. Two small an antibody against this sequence, the RT-PCR product was interfering (si)RNA oligonucleotides were designed to target cloned for expression in bacteria, and the recombinant protein mmg8, and the transfection of either siRNA into cells effectively purified from bacteria was used for immunizing rabbits. The depleted the protein (,85% reduction, Fig. 2A). Cells resulting antibody, designated as 443M, detected a single band of transfected with mmg8-targeting siRNAs exhibited a weak ,150 kDa in HeLa extracts. This is substantially smaller than the background in which Golgi patterns were not detected when expected size of MMG1 (Verde et al., 2001) (Fig. 1A). To cells were labeled with the anti-MMG8 antibody (Fig. 2B), identify this protein band, we immunoprecipitated the protein which confirmed the specificity of anti-MMG8 staining. In these from HeLa cells and excised the band from gels to perform mass- cells, the Golgi ribbons were broken into patches that overlapped spectrometric analysis (Fig. 1B). Tandem mass spectrometry largely with nuclei (Fig. 2B). This Golgi fragmentation could be revealed a total of 13 peptide sequences (supplementary material rescued by the ectopic expression of MMG8 at low levels Fig. S1B). Most of the peptide sequences matched the sequences (Fig. 2B). We also determined that the overexpression of MMG8 from MMG variant 5 (GenBank accession