Acknowledgements: We thank L. Dimitrova-Paternoga (European Molecular Biology Laboratory (EMBL) Heidelberg) for reagents, the EMBL Protein Expression and Purification Core Facility, the EMBL Advanced Light Microscopy Facility, especially M. Lampe, and the EMBL Chemical Biology Core Facility, especially D. Will, for their support. We thank the Bio-SAXS beamline at European Synchrotron Radiation Facility (ESRF) Grenoble, BM29. We thank P. Pernot (ESRF), J. Kieffer (ESRF) and C. Jeffries (EMBL Hamburg) for discussions. S.H. was supported by the EMBL Interdisciplinary Postdoctoral fellowship (EIPOD) Programme under Marie Curie Cofund Actions MSCA-COFUND-FP (grant no. 664726) and Deutsche Forschungsgemeinschaft (DFG)-Forschergruppe 2333 grant (grant no. EP37/4-1) to A.E. The graphics in Fig. 7 were generated by E. Chiang as part of the MRC Laboratory of Molecular Biology’s VisLab, and adapted for use in Fig. 1b. Work in the laboratories of J.H. and A.E. was supported by funding from the DFG via the priority program SPP1935 to J.H. and A.E. (grant nos EP37/3-1 and EP37/3-2) and the EMBL. Work in S.L.B.’s group is supported by the Medical Research Council (MRC), as part of United Kingdom Research and Innovation (also known as UK Research and Innovation; MRC file reference no. MC_U105178790). M.A.M. is supported by a project grant from the Biotechnology and Biological Sciences Research Council (grant no. BB/T00696X/1) awarded to S.L.B. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. For the purpose of the MRC open access policy, the authors have applied a CC-BY public copyright license to any Author Accepted Manuscript version arising.Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. We show through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing competition with dynein until kinesin-1 is activated in the oocyte. Our work reveals a new strategy for ensuring sequential activity of microtubule motors
