AbstractMutations in LMAN1 and MCFD2 cause the combined deficiency of FV and FVIII (F5F8D). LMAN1 and MCFD2 form a protein complex that transport FV and FVIII from the endoplasmic reticulum to the Golgi. Although both proteins are required for the cargo receptor function, little is known about specific roles of LMAN1 and MCFD2 in transporting FV/FVIII. We used different LMAN1 and MCFD2 deficient cell lines to investigate the LMAN1/MCFD2-dependent FV/FVIII secretion pathway. LMAN1 deficiency led to more profound decreases in FV/FVIII secretion in HEK293T and HepG2 cells than in HCT116 cells, suggesting regulation of cargo transport by the LMAN1/MCFD2 pathway varies in different cell types. Using these cell lines, we developed functional assays to accurately assess pathogenicity of recently reported potential LMAN1 and MCFD2 missense mutations. LMAN1 with mutations abolishing carbohydrate binding can still partially rescue FV/FVIII secretion, suggesting that N-glycan binding is not absolutely required for FV/FVIII transport. Surprisingly, overexpression of either WT or mutant MCFD2 is sufficient to rescue FV/FVIII secretion defects in LMAN1 deficient cells. These results suggest that cargo binding and transport are carried out by MCFD2 and that LMAN1 primarily serves as a shuttling carrier of MCFD2. Finally, overexpression of both LMAN1 and MCFD2 does not further increase FV/FVIII secretion, suggesting that the amount of the LMAN1-MCFD2 receptor complex is not a rate-limiting factor in ER-Golgi transport of FV/FVIII. This study provides new insight into the molecular mechanism of F5F8D and intracellular trafficking of FV and FVIII.Key PointsEfficient ER-to-Golgi transport of FV and FVIII requires the LMAN1-MCFD2 cargo receptor complex.MCFD2 functions as a primary interacting partner of FV/FVIII cargo and LMAN1 primarily serves as a shuttling carrier of MCFD2.