Mitochondrial complex I (CI), a large multi-subunit respiratory complex contains two LYR (leucine/tyrosine/arginine) domain-containing subunits, B14 (NDUA6/LYRM6) and B22 (NDUB9/LYRM3). Mitochondrial LYR (LYRM) proteins are soluble matrix-located proteins that have been implicated in diverse functions such as iron-sulphur cluster insertion, OXPHOS complex assembly, and mitoribosome biogenesis. B14 and B22 are unique to other LYRM proteins in that they are integral components of CI. To explore the function of B22, we examined T-DNA insertional knockout and knockdown lines, which displayed a mild growth defect linked to reduced CI activity and abundance. Notably, this defect could not be rescued by complementation with a B22 variant that contained a mutated LYR domain, indicating the domain's critical role in B22's function. Protein interaction assays further revealed that the LYR domain is crucial for B22's interaction with the neighbouring CI subunit, mitochondrial acyl carrier protein SDAP1. Similarly, T-DNA insertional knockdown lines of SDAP1 showed a comparable CI defect, suggesting that the interaction between B22 and SDAP1, mediated by the LYR domain, is important for the function and assembly of CI.