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Atlastin-Mediated Membrane Tethering Is Critical for Cargo Mobility and Exit from the Endoplasmic Reticulum

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Atlastin-Mediated Membrane Tethering Is Critical for Cargo Mobility and Exit from the Endoplasmic Reticulum Atlastin-mediated membrane tethering is critical for cargo mobility and exit from the endoplasmic reticulum Liling Niua,b,1, Tianji Mac,1, Feng Yangc, Bing Yana, Xiao Tangc, Haidi Yind, Qian Wud, Yan Huangc, Zhong-Ping Yaod, Jifeng Wanga, Yusong Guoc,2, and Junjie Hua,2 aNational Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101 Beijing, China; bDepartment of Genetics and Cell Biology, College of Life Sciences, Nankai University, 300071 Tianjin, China; cDivision of Life Science, Hong Kong University of Science and Technology, Hong Kong, China; and dState Key Laboratory of Chemical Biology and Drug Discovery, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China Edited by Peter J. Novick, University of California San Diego, La Jolla, CA, and approved June 5, 2019 (received for review May 17, 2019) Endoplasmic reticulum (ER) membrane junctions are formed by the have revealed that the GTPase domain of ATL forms a dimer dynamin-like GTPase atlastin (ATL). Deletion of ATL results in long upon GTP binding, and GTP hydrolysis causes conformational unbranched ER tubules in cells, and mutation of human ATL1 is changes in the associating 3-helix bundle (3HB) (9, 19, 20). The linked to hereditary spastic paraplegia. Here, we demonstrate that nucleotide-dependent transdimer of ATL tethers apposing COPII formation is drastically decreased in the periphery of ATL- membranes, and the swing of 3HB pulls 2 membranes closer for deleted cells. ER export of cargo proteins becomes defective; ER subsequent fusion. In addition, the TM domains of ATL cluster exit site initiation is not affected, but many of the sites fail to the fusogen in the same membrane and may form intramembrane recruit COPII subunits. The efficiency of cargo packaging into COPII hairpins (21, 22), and an amphipathic helix in the CT promotes vesicles is significantly reduced in cells lacking ATLs, or when the lipid mixing by perturbing the lipid bilayer (21, 23). In vitro studies ER is transiently fragmented. Cargo is less mobile in the ER in the using purified ATL have demonstrated that continuous GTP hy- absence of ATL, but the cargo mobility and COPII formation can be drolysis is necessary for efficient membrane tethering, and most restored by ATL R77A, which is capable of tethering, but not fusion attempts halt at the tethered state (24). When the purified fusing, ER tubules. These findings suggest that the generation of cytosolic domain of ATL is added to an in vitro assembled tubular ER junctions by ATL plays a critical role in maintaining the ER network with Xenopus egg extract, the network is readily dis- CELL BIOLOGY necessary mobility of ER contents to allow efficient packaging of rupted (25), confirming that many junctions are tethered instead of cargo proteins into COPII vesicles. fused. Nevertheless, in cells, the ER membrane system is contin- uous as long as some 3-way junctions are products of actual fusion. endoplasmic reticulum | atlastin | membrane tension | COPII formation | The generation of the tubular ER network is of great physi- protein mobility ological importance. In addition to ATL family-related defects, loss of tubule-forming proteins causes retarded growth in yeast n eukaryotic cells, the endoplasmic reticulum (ER) is mainly (4) and decreases embryonic survival in Caenorhabditis elegans Iresponsible for protein synthesis, lipid synthesis, and calcium (26), and mutations in Rtn2 and REEP1 are linked to HSP. How- storage (1, 2). Morphologically, as one continuous membrane ever, the cellular processes regulated by the tubular ER network are system, the ER consists of cylindrical tubules and cisternal sheets largely unknown. Recent proteomic analysis revealed that 79 pro- (1, 3). A characteristic feature of the ER is the reticular network teins are enriched in the ER tubules of yeast cells (27). Functional of tubules usually seen in the cell periphery. Tubules, which re- categorization of these proteinsimpliesthattubularERmaybe quire high curvature at cross-sections, are shaped by a class of specialized in lipid synthesis, membrane contacts, and stress signaling. integral membrane proteins, the reticulons and REEPs (4, 5), and subsequently connected in the form of 3-way junctions by Significance dynamin-like GTPase atlastin (ATL) (6, 7). Deletion or depletion of ATL in mammalian cells causes long In the early secretory pathway, newly synthesized proteins unbranched ER tubules (6, 8) indicative of a lack of fusion be- undergo folding and modifications and then leave the ER tween tubules. Purified Drosophila ATL mediates vesicle fusion through COPII-coated vesicles. How these processes are co- in vitro in a GTP-dependent manner (7, 9), providing further ordinated and maintained are important but mostly unclear. evidence of its fusogenic activity. The yeast homolog Sey1p and We show here that ATL, a GTPase that connects ER tubules, plant homolog RHD3 have been shown to function similarly (10–12). controls ER protein mobility and regulates cargo packaging and ATL1 mutations are linked to the human disease hereditary coat assembly of COPII vesicles. The tethering and fusion ac- spastic paraplegia (HSP), which is characterized by progressive tivity by ATL likely maintains tension and other necessary pa- spasticity and weakness of the lower limbs due to retrograde de- rameters for COPII formation in ER membranes. These findings generation of corticospinal axons (13, 14). In Arabidopsis thaliana, reveal a role of ER shaping in the early secretory pathway and deletion or mutations of RHD3 are viable but cause prominent provide insight into behaviors of ER exportation. defects in root hair cells (12, 15, 16). Thus, ATL-mediated fusion Author contributions: L.N., Y.G., and J.H. designed research; L.N., T.M., F.Y., X.T., H.Y., between ER membranes plays an important role in cells with long Q.W., and Y.H. performed research; Z.-P.Y. contributed new reagents/analytic tools; L.N., protrusions. However, ATL proteins are expressed ubiquitously T.M., F.Y., B.Y., J.W., Y.G., and J.H. analyzed data; and Y.G. and J.H. wrote the paper. (8), and deletion of RHD3 and either one of its redundant genes The authors declare no conflict of interest. leads to lethality in plants (12), suggesting a more fundamental This article is a PNAS Direct Submission. role of ER membrane fusion. This open access article is distributed under Creative Commons Attribution License 4.0 High eukaryotes possess 3 ATLs, with ATL1 predominantly in (CC BY). the nervous system and ATL2/ATL3 in peripheral tissues (8, 17). 1L.N. and T.M. contributed equally to this work. The enzymatic activity of ATL1 and ATL2 exceeds that of ATL3 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. (18). ATL is composed of N-terminal cytosolic GTPase, followed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. by a helical region, 2 transmembrane (TM) segments, and a C- 1073/pnas.1908409116/-/DCSupplemental. terminal cytosolic tail (CT). Structural and biochemical analyses Published online June 25, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1908409116 PNAS | July 9, 2019 | vol. 116 | no. 28 | 14029–14038 Interestingly, in yeast, COPII vesicles are formed mainly in ER tion, when either Rtn4 or Climp63 was depleted using CRISPR/ tubules (28), and a set of COPII regulators preferentially localize Cas9 or siRNA (SI Appendix, Fig. S2 D and H), the pattern of to the tubular ER (27, 29), suggesting that the tubular ER plays a Sec31A remained unchanged (SI Appendix, Fig. S2 E and I). key role in membrane trafficking. These results collectively suggest that the COPII defects seen COPII-coated vesicles deliver cargo proteins from the ER to here are specifically caused by alterations in ATL activity. the Golgi. The assembly and functions of the COPII coat have As previously reported (8), deletion of ATLs caused some been studied extensively (30–34). ER-anchored GEF protein fragmentation of the Golgi apparatus (SI Appendix, Fig. S1H), Sec12 catalyzes the GTP loading of small GTPase Sar1. GTP- particularly the cis-Golgi network (CGN) marked by GM130. bound Sar1 relocates to the ER and recruits the Sec23/Sec24 However, patterns of β-COP, the coat of COPI, remained similar heterodimer, forming an inner coat. Sec24 often serves as a cargo between wild-type and DKO cells (SI Appendix, Fig. S1I). In adaptor to mediate the packaging of cargo proteins into nascent addition, staining of AP1γ1, the γ-subunit of the AP-1 complex vesicles, and the growing bud further attracts the Sec13/Sec31 localized to the trans-Golgi network (TGN) and endosomes, was heterodimer, forming the outer coat. COPII is assembled at the ER not altered in DKO cells compared with wild type (SI Appendix, exit sites (ERESs) marked by scaffolding protein Sec16. However, Fig. S1J). We also confirmed that the changes seen with Sec31A how the ER morphology interferes with cargo sorting and vesicle apply to Sec24C, a subunit of the COPII inner coat, when these formation processes at ERESs remains unclear. coats were costained (SI Appendix,Fig.S1G). These results Here, we show that deletion of ATLs causes defects in COPII suggest that defects caused by ATL deletion are specific to the formation. Lack of ATL results in delayed cargo exit and coat entire COPII coat. assembly. We also show that COPII defects are likely due to al- Next, we tested whether the reduction in COPII puncta is tered cargo mobility and ATL-mediated membrane tethering, but caused by changes in the levels of coat proteins. Compared with not fusion. These findings provide important insight into the wild-type COS-7 cells, no difference in expression was detected physiological role of the tubular ER network.
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