Search

Chloroplast proteins are imported via the translocon at the outer chloroplast membrane (TOC)-translocon at the inner chloroplast membrane (TIC) supercomplex, driven by an ATPase motor. The Ycf2-FtsHi complex has been identified as the chloroplast import motor. However, its assembly and cooperation with the TIC complex during preprotein translocation remain unclear. Here, we present the structures of the Ycf2-FtsHi and TIC complexes from Arabidopsis and an ultracomplex formed between them from Pisum. The Ycf2-FtsHi structure reveals a heterohexameric AAA+ ATPase motor module with characteristic features. Four previously uncharacterized components of Ycf2-FtsHi were identified, which aid in complex assembly and anchoring of the motor module at a tilted angle relative to the membrane. When considering the structures of the TIC complex and the TIC-Ycf2-FtsHi ultracomplex together, it becomes evident that the tilted motor module of Ycf2-FtsHi enables its close contact with the TIC complex, thereby facilitating efficient preprotein translocation. Our study provides valuable structural insights into the chloroplast protein import process in land plants. [Display omitted] • Isolation of native AtYcf2-FtsHi, AtTIC, and PsTIC-Ycf2-FtsHi ultracomplex • The composition and assembly of Arabidopsis Ycf2-FtsHi and TIC complexes are revealed • Ycf2-FtsHi poses a tilted heterohexameric motor module staying close to TIC • An ultracomplex structure unveils the functional cooperation between TIC and Ycf2-FtsHi Chloroplast protein import through the TOC-TIC is driven by an essential ATPase motor. The structure of a translocation intermediate ultracomplex between TIC and Ycf2-FtsHi from Pisum , along with the individual structures of the two complexes from Arabidopsis , confirms Ycf2-FtsHi as the import motor and provides comprehensive insights into the chloroplast protein import process. [ABSTRACT FROM AUTHOR]

The TOC and TIC complexes are essential translocons that facilitate the import of the nuclear genome-encoded preproteins across the two envelope membranes of chloroplast, but their exact molecular identities and assembly remain unclear. Here, we report a cryoelectron microscopy structure of TOC-TIC supercomplex from Chlamydomonas , containing a total of 14 identified components. The preprotein-conducting pore of TOC is a hybrid β-barrel co-assembled by Toc120 and Toc75, while the potential translocation path of TIC is formed by transmembrane helices from Tic20 and YlmG, rather than a classic model of Tic110. A rigid intermembrane space (IMS) scaffold bridges two chloroplast membranes, and a large hydrophilic cleft on the IMS scaffold connects TOC and TIC, forming a pathway for preprotein translocation. Our study provides structural insights into the TOC-TIC supercomplex composition, assembly, and preprotein translocation mechanism, and lays a foundation to interpret the evolutionary conservation and diversity of this fundamental translocon machinery. [Display omitted] • Chlamydomonas TOC-TIC supercomplex contains a total of 14 identified components • Toc120 and Toc75 form a hybrid β-barrel pore of TOC for preprotein conducting • Tic20 and YlmG constitute a potential translocation path of TIC with lateral opening • An IMS scaffold bridges the TOC and TIC to form a preprotein translocation pathway Cryoelectron microscopy structure of TOC-TIC supercomplex from Chlamydomonas reveals its composition, assembly, and the mechanism for chloroplast preprotein translocation. [ABSTRACT FROM AUTHOR]

The protein import motor in chloroplasts plays a pivotal role in their biogenesis and homeostasis by driving the translocation of preproteins into chloroplasts. While the Ycf2-FtsHi complex serves as the import motor in land plants, its evolutionary conservation, specialization, and mechanisms across photosynthetic organisms are largely unexplored. Here, we isolated and determined the cryogenic electron microscopy (cryo-EM) structures of the native Ycf2-FtsHi complex from Chlamydomonas reinhardtii , uncovering a complex composed of up to 19 subunits, including multiple green-algae-specific components. The heterohexameric AAA+ ATPase motor module is tilted, potentially facilitating preprotein handover from the translocon at the inner chloroplast membrane (TIC) complex. Preprotein interacts with Ycf2-FtsHi and enhances its ATPase activity in vitro. Integrating Ycf2-FtsHi and translocon at the outer chloroplast membrane (TOC)-TIC supercomplex structures reveals insights into their physical and functional interplay during preprotein translocation. By comparing these findings with those from land plants, our study establishes a structural foundation for understanding the assembly, function, evolutionary conservation, and diversity of chloroplast protein import motors. [Display omitted] • Structure of the Chlamydomonas Ycf2-FtsHi complex reveals its composition and assembly • ATP binding induces a conformational change in the ATPase domain of Ctap1 • Preprotein interacts with Ycf2-FtsHi and enhances its ATPase activity in vitro • Ycf2-FtsHi exhibits evolutionary diversity between green algae and land plants The Ycf2-FtsHi complex functions as a TIC complex-associated ATPase motor in chloroplast protein import. Cryo-EM structure of the native Chlamydomonas Ycf2-FtsHi complex reveals up to 19 subunits, including conserved heterohexameric motor components and multiple green-algae-specific components, which offers evolutionary insights into the conservation and diversity of the chloroplast import motor across species. [ABSTRACT FROM AUTHOR]