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The Cellulose Synthases Are Cargo of the TPLATE Adaptor Complex

2017; Elsevier BV; Volume: 11; Issue: 2 Linguagem: Inglês

10.1016/j.molp.2017.11.012

1674-2052

Clara Sánchez‐Rodríguez, Yanyun Shi, Christopher Kesten, Dongmei Zhang, Gloria Sáncho-Andrés, Alexander Ivakov, Edwin R. Lampugnani, Kamil Skłodowski, Masaru Fujimoto, Akihiko Nakano, Antony Bacic, Ian S. Wallace, Takashi Ueda, Daniël Van Damme, Yihua Zhou, Staffan Persson,

Plant nutrient uptake and metabolism

Clathrin-mediated endocytosis (CME) is an evolutionary conserved mechanism by which plasma membrane (PM)-based cargo proteins are recognized by adaptor protein complexes and internalized. Apart from the canonical adaptor complex, AP-2, plant cells rely on the TPLATE complex (TPC) to execute CME (Gadeyne et al., 2014Gadeyne A. Sanchez-Rodriguez C. Vanneste S. Di Rubbo S. Zauber H. Vanneste K. Van Leene J. De Winne N. Eeckhout D. Persiau G. et al.The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.Cell. 2014; 156: 691-704Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The TPC is an octameric protein complex, consisting of TPLATE, TASH3, LOLITA, TWD40-1, TWD40-2, TML, AtEH1, and AtEH2 (Gadeyne et al., 2014Gadeyne A. Sanchez-Rodriguez C. Vanneste S. Di Rubbo S. Zauber H. Vanneste K. Van Leene J. De Winne N. Eeckhout D. Persiau G. et al.The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.Cell. 2014; 156: 691-704Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). As the complex components are not conserved in yeast and animal cells, CME initiation appears to be regulated differently in plants (Hirst et al., 2014Hirst J. Schlacht A. Norcott J.P. Traynor D. Bloomfield G. Antrobus R. Kay R.R. Dacks J.B. Robinson M.S. Characterization of TSET, an ancient and widespread membrane trafficking complex.Elife. 2014; 3: e02866Crossref PubMed Google Scholar). This raises important evolutionary questions concerning CME and cargo recognition across eukaryotic Kingdoms (Zhang et al., 2015Zhang Y. Persson S. Hirst J. Robinson M.S. van Damme D. Sanchez-Rodriguez C. Change your TPLATE, change your fate: plant CME and beyond.Trends Plant Sci. 2015; 20: 41-48Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Based on in silico analysis, TASH3, TPLATE, TML, and LOLITA might have functions related to AP-2A, AP-2B, AP-2M, and AP-2S, respectively (Hirst et al., 2014Hirst J. Schlacht A. Norcott J.P. Traynor D. Bloomfield G. Antrobus R. Kay R.R. Dacks J.B. Robinson M.S. Characterization of TSET, an ancient and widespread membrane trafficking complex.Elife. 2014; 3: e02866Crossref PubMed Google Scholar, Zhang et al., 2015Zhang Y. Persson S. Hirst J. Robinson M.S. van Damme D. Sanchez-Rodriguez C. Change your TPLATE, change your fate: plant CME and beyond.Trends Plant Sci. 2015; 20: 41-48Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), which may include cargo recognition and clathrin recruitment to the PM. Indeed, similar to Arabidopsis AP2M and AP2S (Fan et al., 2013Fan L. Hao H. Xue Y. Zhang L. Song K. Ding Z. Botella M.A. Wang H. Lin J. Dynamic analysis of Arabidopsis AP2 sigma subunit reveals a key role in clathrin-mediated endocytosis and plant development.Development. 2013; 140: 3826-3837Crossref PubMed Scopus (106) Google Scholar), TPLATE and TML are required for clathrin recruitment to the PM, even after AP-2 depletion (Wang et al., 2016Wang C. Hu T. Yan X. Meng T. Wang Y. Wang Q. Zhang X. Gu Y. Sanchez-Rodriguez C. Gadeyne A. et al.Differential regulation of clathrin and its adaptor proteins during membrane recruitment for endocytosis.Plant Physiol. 2016; 171: 215-229Crossref PubMed Scopus (45) Google Scholar). Therefore, we hypothesized that these proteins are involved in cargo recognition in plants. Loss of TPC function results in male sterility (Gadeyne et al., 2014Gadeyne A. Sanchez-Rodriguez C. Vanneste S. Di Rubbo S. Zauber H. Vanneste K. Van Leene J. De Winne N. Eeckhout D. Persiau G. et al.The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.Cell. 2014; 156: 691-704Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), similar to loss-of-function mutants of cellulose synthesis (Persson et al., 2007Persson S. Paredez A. Carroll A. Palsdottir H. Doblin M. Poindexter P. Khitrov N. Auer M. Somerville C.R. Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2007; 104: 15566-15571Crossref PubMed Scopus (413) Google Scholar). CME has been reported to mediate internalization of cellulose synthase (CESA) complexes (CSCs) in elongating hypocotyl cells (Bashline et al., 2013Bashline L. Li S. Anderson C.T. Lei L. Gu Y. The endocytosis of cellulose synthase in Arabidopsis is dependent on mu2, a clathrin-mediated endocytosis adaptin.Plant Physiol. 2013; 163: 150-160Crossref PubMed Scopus (118) Google Scholar, Bashline et al., 2015Bashline L. Li S. Zhu X. Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.Proc. Natl. Acad. Sci. USA. 2015; 112: 12870-12875Crossref PubMed Scopus (61) Google Scholar), which is also supported by live-cell imaging data (Supplemental Figure 1; Miart et al., 2014Miart F. Desprez T. Biot E. Morin H. Belcram K. Hofte H. Gonneau M. Vernhettes S. Spatio-temporal analysis of cellulose synthesis during cell plate formation in Arabidopsis.Plant J. 2014; 77: 71-84Crossref PubMed Scopus (73) Google Scholar). Since cellulose synthesis is essential for plant growth and the CSCs are unique to plants, it might be expected that the CESAs are TPC cargo, which could explain why the TPC is maintained in plants but not in animals and yeasts. To test this, we performed co-immunoprecipitation experiments, which revealed that CESA6, a component of the primary wall CSC, interacts with both TML and TPLATE (Figure 1A, upper panel). We corroborated this finding by bimolecular fluorescence complementation (BiFC) between the Arabidopsis primary wall CESAs, i.e., CESA1, -3, and -6 and TPLATE (Figure 1A bottom panel and Supplemental Figure 2). To confirm that the detected fluorescent signals were not due to either overexpression or random collisions of the split FP halves, we co-expressed each of the primary CESAs with AtEH1, another subunit of the TPC. Here, we did not observe any detectable fluorescent signal from the BiFC assays (Figure 1A bottom panel and Supplemental Figure 2A and 2C), confirming that the TPLATE–CESA interactions are specific in our BiFC system and suggesting a lack of direct interaction between AtEH1 and the CESAs, while being connected through TPLATE. Notably, the CESA–TPLATE interactions correlated with a change in CESA localization patterns; from a homogeneous and diffuse distribution to a speckled pattern (Figure 1A bottom panel and Supplemental Figure 2A), possibly indicating internalized CSCs. To test this hypothesis, we repeated the BiFC assay between CESA6 and TPLATE in the presence of the clathrin marker CLC2-mCh (Supplemental Figure 2B and 2D). We found that the BiFC signal of the CESA–TPLATE co-localized with the clathrin marker CLC2-mCh (Supplemental Figure 2B). As expected from the multiple roles of clathrin, we also observed mCherry fluorescence at cellular compartments not marked by TPLATE–CESA interactions. Defects in CESA internalization are likely to result in severe cellulose deficiency. Accordingly, lines altered in TML expression, pESTR:amiR-TML (Gadeyne et al., 2014Gadeyne A. Sanchez-Rodriguez C. Vanneste S. Di Rubbo S. Zauber H. Vanneste K. Van Leene J. De Winne N. Eeckhout D. Persiau G. et al.The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.Cell. 2014; 156: 691-704Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), displayed defects in hypocotyl and root elongation (Figure 1B upper panels), concomitant with cell swelling (Supplemental Figure 3C), similar to what is found in seedlings impaired in primary wall cellulose synthesis (McFarlane et al., 2014McFarlane H.E. Doring A. Persson S. The cell biology of cellulose synthesis.Annu. Rev. Plant Biol. 2014; 65: 69-94Crossref PubMed Scopus (349) Google Scholar). Furthermore, we observed a strong reduction in cellulose content and an increase in sugars corresponding to non-cellulosic polysaccharides, mainly those of pectin-related monosaccharides (e.g., uronic acids) compared with control seedlings (Figure 1B bottom panel and Supplemental Table 1). Plants impaired in two other main steps of CME: vesicle assembly by clathrin triskelia (pINTAM ≫ RFP-HUB) and vesicle scission (drp1a-2/rsw9-2), showed similar phenotypes and cell-wall composition to the amiR:TML line (Supplemental Figure 3 and Supplemental Table 1). Interestingly, a null mutant for a subunit of the other plant early adaptor complex, the canonical AP-2 (ap2m-1), showed increase in hypocotyl length, did not display defects in cell swelling, and its sugar composition was comparable with wild-type seedlings (Supplemental Figure 3, Supplemental Table 1; Bashline et al., 2015Bashline L. Li S. Zhu X. Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.Proc. Natl. Acad. Sci. USA. 2015; 112: 12870-12875Crossref PubMed Scopus (61) Google Scholar). Defects in CESA internalization should have an impact on CESA dynamics at the PM. Live-cell imaging revealed an increase in FP-CESA6 density at the PM in amiR-TML seedlings compared with their control (0.9 ± 0.1 foci/μm2 in control cells versus 1.6 ± 0.1 foci/μm2 in amiR-TML cells; Figure 1C left and upper right panels). The increase in FP-CESA6 density could be due to either an enhanced delivery or a reduced internalization rate of the CSCs to and from the PM, respectively. To differentiate between these processes, we calculated the CESA delivery rate (Gutierrez et al., 2009Gutierrez R. Lindeboom J.J. Paredez A.R. Emons A.M. Ehrhardt D.W. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.Nat. Cell Biol. 2009; 11: 797-806Crossref PubMed Scopus (506) Google Scholar) and found no differences in CESA delivery rate to the PM between the lines (Figure 1C upper right panel). The increased CESA6 density at the PM should therefore reflect a reduction in the internalization rate of CSC when TML activity is impaired. Moreover, the reduction of CESA6 internalization correlated with an increased amount of slow-moving CESA particles at the PM (indicated by less tilted traces in kymographs; Figure 1C left panel). The drp1a-2 mutant showed similar CSC homeostasis at the PM as observed for amiR-TML; i.e., increased CESA6 density and slow-moving particles (Supplemental Figure 4). As CESA movement is related to its activity, this explains the reduced cellulose content in the TML and DRP1A knockdown/knockout lines. As reported previously (Bashline et al., 2015Bashline L. Li S. Zhu X. Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.Proc. Natl. Acad. Sci. USA. 2015; 112: 12870-12875Crossref PubMed Scopus (61) Google Scholar), we did not observe any differences in the CSC speed at the PM between ap2m-1 and wild-type cells (Supplemental Figure 4) and nor did we observe any significant differences in FP-CESA6 density at the PM in ap2m-1 compared with wild-type (Supplemental Figure 4B). While these data contrast with previous reports (Bashline et al., 2013Bashline L. Li S. Anderson C.T. Lei L. Gu Y. The endocytosis of cellulose synthase in Arabidopsis is dependent on mu2, a clathrin-mediated endocytosis adaptin.Plant Physiol. 2013; 163: 150-160Crossref PubMed Scopus (118) Google Scholar, Bashline et al., 2015Bashline L. Li S. Zhu X. Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.Proc. Natl. Acad. Sci. USA. 2015; 112: 12870-12875Crossref PubMed Scopus (61) Google Scholar), they do explain the absence of cellulose and anisotropic cell growth reduction in the ap2m-1 mutant compared with the wild-type (Supplemental Figure 4; Bashline et al., 2015Bashline L. Li S. Zhu X. Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.Proc. Natl. Acad. Sci. USA. 2015; 112: 12870-12875Crossref PubMed Scopus (61) Google Scholar). Our results therefore indicate that the TPC is key in the regulation of CSC trafficking and, hence, cellulose synthesis in Arabidopsis seedlings. To explore whether CESAs are also internalized via TPC-related CME in other plant species, we performed split-luciferase assays combining the rice TPC proteins OsTML and OsTPLATE2, with OsCESA8 and OsCESA4, which are primary and secondary wall rice CESAs, respectively. We could detect clear luciferase signals from these assays, which indicate that the rice CSCs also interact with TPC components (Figure 1D). In addition, we performed immuno-precipitation (IP) using an OsCESA4 antibody coupled with mass spectrometry (MS) to identify OsCESA4 interactors in rice plants. Notably, we found several TPC subunits, including OsTPLATE2, OsTML, OsTWD40-1, OsEH2, OsTASH3, and OsDRP2B/BC3, as well as other cellulose-related proteins in the pellet (Supplemental Table 2). Our MS results did not identify AP-2 subunits in the pull-downs of any of our IP replicates. Hence, CSCs might also be CME cargo recruited by the TPC early adaptor in rice. In vivo evidence for cargo-CME co-internalization and re-localization of cargo by the CME components via BiFC has not been reported in plants. In addition, we were able to identify a plant CME cargo by IP-MS, which is rare in plant biology. Hence, our results provide a foundation for further elaborations of CME-mediated events in plant cells. Our in vivo interaction data between TPLATE and TML with CSC, in both Arabidopsis and rice, indicate that TPLATE and TML might recognize the CSC for its internalization. TML has a muHD domain that is also present in APμ subunits (e.g., AP-2M in the AP-2 complex) and in the muniscins (Hirst et al., 2014Hirst J. Schlacht A. Norcott J.P. Traynor D. Bloomfield G. Antrobus R. Kay R.R. Dacks J.B. Robinson M.S. Characterization of TSET, an ancient and widespread membrane trafficking complex.Elife. 2014; 3: e02866Crossref PubMed Google Scholar, Zhang et al., 2015Zhang Y. Persson S. Hirst J. Robinson M.S. van Damme D. Sanchez-Rodriguez C. Change your TPLATE, change your fate: plant CME and beyond.Trends Plant Sci. 2015; 20: 41-48Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Thus, it might be anticipated that TML can recognize cargo. Notably, some protein domains suggest a relationship between TPLATE and AP-2B, which does not include any cargo-recognition motif. Our in vivo interaction results between TPLATE and CSC suggest that the TPLATE subunit of the TPC may have a cargo-binding capacity in plant cells. Our results indicate that the TPC is the main early adaptor that recognizes CSC for its internalization via clathrin. The data presented here are in agreement with the fact that the TWD40-2 subunit of the TPC contributes to cellulose synthesis (Bashline et al., 2015Bashline L. Li S. Zhu X. Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.Proc. Natl. Acad. Sci. USA. 2015; 112: 12870-12875Crossref PubMed Scopus (61) Google Scholar), supporting a prominent role of the TPC in this process. AP2-M has been shown to bind to the central cytosolic domain of the primary CESAs (Bashline et al., 2013Bashline L. Li S. Anderson C.T. Lei L. Gu Y. The endocytosis of cellulose synthase in Arabidopsis is dependent on mu2, a clathrin-mediated endocytosis adaptin.Plant Physiol. 2013; 163: 150-160Crossref PubMed Scopus (118) Google Scholar), which contain putative AP-2M binding domains (YxxΦ) in a yeast assay. Therefore, the CSC might be recognized by both plant TPC and AP-2 early adaptors, possibly at different regions of its cytosolic domains. Notably, TPC and AP-2 co-localize in approximately 50% of all CME events, suggesting a complementary role of these CME adaptors (Gadeyne et al., 2014Gadeyne A. Sanchez-Rodriguez C. Vanneste S. Di Rubbo S. Zauber H. Vanneste K. Van Leene J. De Winne N. Eeckhout D. Persiau G. et al.The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.Cell. 2014; 156: 691-704Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), plausibly relating to particular cargo or certain growth circumstances. This might be the case under certain stress conditions, such as those experienced by the plant when the TPC is not fully active (Barth and Holstein, 2004Barth M. Holstein S.E. Identification and functional characterization of Arabidopsis AP180, a binding partner of plant alphaC-adaptin.J. Cell Sci. 2004; 117: 2051-2062Crossref PubMed Scopus (56) Google Scholar, Bashline et al., 2015Bashline L. Li S. Zhu X. Gu Y. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.Proc. Natl. Acad. Sci. USA. 2015; 112: 12870-12875Crossref PubMed Scopus (61) Google Scholar). In summary, although it has been shown that several cargo proteins showed defective internalization upon depletion of TPC subunits (Gadeyne et al., 2014Gadeyne A. Sanchez-Rodriguez C. Vanneste S. Di Rubbo S. Zauber H. Vanneste K. Van Leene J. De Winne N. Eeckhout D. Persiau G. et al.The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants.Cell. 2014; 156: 691-704Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), our study discovered the CSC as the first identified TPC–cargo interaction in seed plants, recognized by TML and TPLATE subunits, providing new insights into the evolution of cargo recognition in plant CME. This work was supported by the Max-Planck Gesellschaft, the Deutsche Forschungsgemeinschaft, the National Natural Science Foundation of China (grants 31530051), the Swiss Federal Institute of Technology of Zurich (ETH-Z), the Swiss National Foundation (SNF 2-77212-15), the University of Melbourne, the Australian Research Council (CE1101007, FT160100218, DP110100410), the Ministry of Education, Culture, Sports, Science, and Technology of Japan (24114003, 15H04382, and 17K19412), the European Research Council (ERC grant 682436), the IRRTF-RNC (no. 501892) and a USA National Science Foundation CAREER Award.