FIG 4.

MceA is farnesylated in HeLa cells, but this does not influence mitochondrial localization. (A) HeLa cells were transfected with pFLAG-MceA for 24 h before being treated with mevastatin (10 μM), L-744,832 (5 μg/ml), or GGTI-298 (1 μM) for 16 h. Cell lysates were collected, separated by SDS-PAGE, and transferred to a nitrocellulose membrane before being probed with anti-FLAG. Ectopically expressed ^3×FLAGMceA forms two distinct bands when detected with anti-FLAG. The lower band represents a prenylated form of ^3×FLAGMceA, as both the prenylation inhibitor mevastatin and the farnesyltransferase inhibitor L-744,832, but not the geranylgeranyltransferase I inhibitor GGTI-298, abolished the presence of this form of ^3×FLAGMceA. Prenylation was confirmed by the absence of the lower band when ^3×FLAGMceA C260A or ^3×FLAGMceA C260S was ectopically expressed. (B) MceA prenylation occurs during infection, as the unmodified ^3×FLAGMceA C260S expressed and translocated by Coxiella is observed as a band higher than the band for translocated ^3×FLAGMceA. Lysate was collected from infected HeLa cells, and proteins were detected with anti-FLAG. (C to E) Host modification does not alter the mitochondrial localization of MceA. HeLa cells were persistently infected with Coxiella expressing ^3×FLAGMceA and treated with either L-744,832 (C) or GGTI-298 (D) for 16 h before being stained with MitoTracker Red and anti-FLAG. Under both treatment conditions, MceA still demonstrated specific punctate mitochondrial localization. (E) Similarly, unmodified ^3×FLAGMceA C260S expressed by Coxiella was consistently observed in the same pattern. *, CCVs. Bars, 10 μm.