Validation of fmkj1-Interacting Proteins Using the Matchmaker Gold Yeast Two-Hybrid (Y2H) System

The Matchmaker Gold Yeast Two-Hybrid (Y2H) System (Clontech, Palo Alto, CA, USA) was employed to validate fmkj1-interacting proteins identified from IP-LC-MS/MS. pGBKT7 was utilized as the bait vector for fmkj1 (without signal peptide, SP) while pGADT7 was used as the prey vector for fmpc1 (without SP). Various truncated fmkj1 mutants with deletions in the N-terminal region were generated via PCR amplification and subcloned into pGBKT7. Co-transformed yeast cells were assessed for growth on synthetic dropout (SD)/-Trp-Leu plates and SD/-Trp-Leu-HisAde plates containing X-galactosidase (X-gal) and aureobasidin A, in accordance with the manufacturer's instructions.