The plastid stroma-localized chaperone HSP90C is essential for maintaining chloroplast proteostasis and facilitating protein translocation. Prior research has established HSP90C's imperative role in the SEC translocase-dependent transport of the photosystem II subunit PsbO1 and its interaction with the SEC1 translocase motor protein SecA1. However, the exact mechanism of this interaction remains to be explored. In this study, we delineated the interactional mode of HSP90C and SecA1 with the model client protein. Yeast two-hybrid and in vitro ATPase activity analyses with purified proteins revealed PsbO1 may bind to HSP90C at multiple sites, including the DPW motif within the C-terminal extension (CTE) region, suggesting a possible client-loading mechanism unique to plastid orthologs. We also confirmed that glycine-646 is important in mediating substrate interaction, though it conferred a much weaker binding than the CTE region, thereby elucidating a critical role for the amino acid whose mutation resulted in visible plant phenotypes. Our in vitro biochemical assays also demonstrated that the stromal intermediate form of PsbO1 with the thylakoid signal peptide (tSP) significantly enhanced SecA1 ATPase activity, suggesting a preferential binding to the motor protein. On the other hand, the mature domain of the PsbO1, excluding the tSP sequence, inhibited HSP90C ATPase activity. We also observed the HSP90C-PsbO1-SecA1 ternary complex was stabilized by the presence of the client tSP. This work therefore provides new insights into the functional mechanisms of HSP90C and its contribution to chloroplast stromal protein stabilization and thylakoid protein transport.