Seung-Yeol Park^1,2,3, Jia-Shu Yang^1,2, Zhen Li⁴, Pan Deng^5,6, Xiaohong Zhu⁴, David Young^1,2, Maria Ericsson⁷, Ruben L.H. Andringa⁸, Adriaan J. Minnaard⁸, Chunmei Zhu^9,10, Fei Sun^9,10,11, D. Branch Moody^1,2, Andrew J. Morris^5,6, Jun Fan^4,12 & Victor W. Hsu^1,2,*

¹ Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, 60 Fenwood Road, Boston, MA 02115, USA. ² Department of Medicine, Harvard Medical School, Boston, MA 02115, USA. ³ Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk 37673, Republic of Korea. ⁴ Department of Materials Science and Engineering, City University of Hong Kong, Tat Chee Avenue, 999077 Hong Kong, China. ⁵ Division of Cardiovascular Medicine, Department of Medicine, University of Kentucky, 741S Limestone, Lexington, KY 40536, USA. ⁶ Lexington Veterans Affairs Medical Center, Lexington, KY 40536, USA. ⁷ Department of Cell Biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA. ⁸ Stratingh Institute for Chemistry, University of Groningen, 9801 MX Groningen, The Netherlands. ⁹ National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, ¹⁵ Datun Road, Chaoyang District, 100101 Beijing, China. ¹⁰ University of Chinese Academy of Sciences, 100101 Beijing, China. ¹¹ Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, 100101 Beijing, China. ¹² City University of Hong Kong, Shenzhen Research Institute, 518057 Shenzhen, China.

^*Correspondence and requests for materials should be addressed to V.W.H.

Studies on vesicle formation by the Coat Protein I (COPI) complex have contributed to a basic understanding of how vesicular transport is initiated. Phosphatidic acid (PA) and diacylglycerol (DAG) have been found previously to be required for the fission stage of COPI vesicle formation. Here, we find that PA with varying lipid geometry can all promote early fission, but only PA with shortened acyl chains promotes late fission. Moreover, diacylglycerol (DAG) acts after PA in late fission, with this role of DAG also requiring shorter acyl chains. Further highlighting the importance of the short-chain lipid geometry for late fission, we find that shorter forms of PA and DAG promote the vesiculation ability of COPI fission factors. These findings advance a general understanding of how lipid geometry contributes to membrane deformation for vesicle fission, and also how proteins and lipids coordinate their actions in driving this process.