In studying the role of P-gp in drug resistance, pharmacokinetics, or normal cell physiology, it is important to have a correct understanding of the basic cellular biology of P-gp. Following two recent reports that P-gp is present in mitochondria [14
], we performed detailed studies to determine whether P-gp does, indeed, localize to mitochondria, and whether this location should be considered when studying the role of P-gp in cellular processes or when studying transport across mitochondrial membranes.
We found that purification of mitochondria by differential centrifugation alone is not adequate for mitochondrial-specific studies. The mitochondrial fractions used in the previous studies which assigned P-gp to mitochondrial membranes were contaminated with other cellular membranes [14
]. Their mitochondrial fractions corresponded to our “crude” mitochondrial fraction; our crude mitochondrial fraction was generated by centrifugation at 5000 × g
for 10 min, while the mitochondrial fractions presented in the other reports were collected by centrifugation at 10,000 × g
for either 10 [14
] or 20 [15
] minutes. Thus, any contaminates present in our crude mitochondrial fraction, produced at a lower g
-force, should also be present in the mitochondrial fractions used in the cited studies. Our crude mitochondrial fraction contained not only mitochondria, but other cellular membranes, including P-gp-rich plasma membranes.
In agreement with the previous reports, we identified P-gp in this mitochondrial-enriched crude fraction, but took further steps to determine if the P-gp present in this fraction was located in mitochondrial membranes. Following purification on an iodixanol gradient, the fraction reacted only with anti-complex III, and no other membrane specific antibodies, indicating a successful separation of mitochondrial membranes from other cellular membranes. This fraction did not react with the anti-P-gp antibody C219, indicating that P-gp was not present in the mitochondria of either KB-V1 or MCF-7ADR cells. Therefore, the assignment of P-gp to mitochondrial membranes by the previous reports was incorrect.
To properly assign the P-gp present in the crude mitochondrial fraction to a cellular membrane, we took a fraction of the crude mitochondrial samples, and using a sucrose gradient, purified the plasma membranes from the other cellular membranes, as indicated by reaction of the resulting fraction with only the anti-integrinα2/VLA-2α antibody and no other cellular membrane markers. Reaction of this fraction with the anti-P-gp antibody C219 indicated that P-gp was present in the plasma membranes of both KB-V1 and MCF-7ADR cells.
Both previous reports [14
] used confocal microscopy to confirm the localization of P-gp to mitochondria. While those authors claim P-gp labeling overlaps with mitochondria-specific labels, a lack of P-gp labeling at the plasma membrane, where P-gp is known to reside [5
], may indicate technical problems with those labeling studies. Additionally, interpretation of data obtained with the C219 monoclonal antibody using confocal microscopy [15
] needs to be performed with caution, as C219 recognizes several different proteins, including MDR3 and myosin [23
], and when not coupled with correct molecular weight identification, as done with Western blot, can lead to false positive results.
Using confocal microscopy, we confirmed that P-gp is not present in the mitochondria of either KB-V1 or MCF-7ADR
cells. Following fixation/permeabilization, reaction of the P-gp specific antibody 17F9-FITC clearly labels the plasma membrane, as expected, indicating reaction with P-gp. No intracellular labeling is observed with this antibody, but staining with DAPI indicates the cells are permeable to the 17F9 antibody; thus if any intracellular P-gp epitopes are present (including mitochondrial or nuclear), they should be accessible to 17F9-FITC. Additionally, no overlap between the mitochondria-specific label Mitotracker and 17F9-FITC was observed in either KB-V1 or MCF7ADR
cells, confirming that P-gp is not present in the mitochondria of these cell lines. These results confirm all of the original immunolocalization studies on P-gp, none of which suggested mitochondrial localization [23
While the exact cell lines used in our study and the two previous reports (P5 hepatocellular carcinoma [15
] or K562 [14
]) differ, in each case the up-regulation of P-gp resulted from selection for doxorubicin (adriamycin) resistance. We also added a cell line selected for resistance to vinblastine. Regardless of the cell line (KB-3-1 or MCF-7) or the drug (doxorubicin or vinblastine) used during selection and up-regulation of P-gp, we did not observe a variation in the cellular localization of P-gp. Furthermore, we believe the assignment of P-gp to mitochondrial membranes in the previous reports was not due to the cell lines used, but due to the fact that purified mitochondria were never obtained.
We believe the P-gp-mediated mitochondrial transport reported in the previous studies [14
] is an artifact of the plasma membrane contamination in the mitochondrial fractions. Purified mitochondria were not isolated in the previous studies, thus the functional assays carried out were with mixtures of membranes. Transport data presented in these previous studies very likely represents transport across plasma membrane vesicles present in the crude mitochondrial fraction. Furthermore, upon vesicle formation, there are two possible protein orientations for a membrane protein: right-side out or wrong-side out. This variation in protein orientation may account for the observed inward transport of one study [14
], and outward transport [15
] of the other.
In conclusion, P-gp is not present in mitochondrial membranes. Therefore, the movement of drugs, or other substances, into or out of the mitochondria is not P-gp-dependent, and the numerous effects of P-gp in the cell are not due to a subcellular localization in mitochondria.