G1P3/IFI6是一种干扰素刺激基因，具有抗凋亡、促转移和抗病毒功能。尽管具有多功能性，G1P3的亚细胞定位仍不清楚。本研究使用生化和共聚焦显微镜技术，确定了G1P3在内质网膜系统和乳腺癌细胞线粒体中的定位。在细胞分离研究中，干扰素诱导的内源性和稳定表达的G1P3与亲和分离的线粒体共沉积。蛋白酶保护实验的结果表明，约24%的线粒体G1P3位于线粒体内。与之相符，表达表位标记的G1P3的共聚焦显微镜研究（MCF-7/G1P3-FLAG）确定其在线粒体（约38%）以及内质网（ER）、转高尔基体（TGN）、溶酶体和RAB5阳性（RAB5+）内体中的定位。这些结果表明G1P3从TGN转运至内溶酶体。G1P3和RAB5都被认为通过线粒体稳定性提高了细胞的抗凋亡能力。因此，测试了G1P3对RAB5在线粒体中的定位的影响。与空载体对照相比，在MCF-7/G1P3-FLAG表达细胞中，RAB5与线粒体的共现率增加了1.5倍（p ≤ .005）。综上所述，我们的结果表明G1P3在促进RAB5阳性内体与线粒体的结合中起到作用，并揭示了G1P3诱导的癌细胞存活的另一机制。©2023国际细胞生物学联合会

G1P3/IFI6 is an interferon stimulated gene with antiapoptotic, prometastatic, and antiviral functions. Despite its pleiotropic functions, subcellular localization of G1P3 remains unclear. Using biochemical- and confocal microscopic approaches, this study identified the localization of G1P3 in organelles of the endomembrane system and in the mitochondria of breast cancer cells. In cell fractionation studies, both interferon-induced endogenous- and stably expressed G1P3 cofractionated with affinity-isolated mitochondria. Results of the protease protection assay have suggested that ~24% of mitochondrial G1P3 resides within the mitochondria. Conforming to this, confocal microscopy studies of cells stably expressing epitope-tagged G1P3 (MCF-7/G1P3-FLAG), identified its localization in mitochondria (~38%) as well as in ER, trans-Golgi network (TGN), lysosomes, and in RAB5 positive (RAB5+ ) endosomes. These results suggested the trafficking of G1P3 from TGN into endolysosomes. Both G1P3 and RAB5 were known to confer apoptosis resistance through mitochondrial stabilization. Therefore, the effects of G1P3 on the localization of RAB5 in mitochondria were tested. Compared to vector control, the co-occurrence of RAB5 with the mitochondria was increased by 1.5-fold in MCF-7/G1P3-FLAG expressing cells (p ≤ .005). Taken together, our results demonstrate a role for G1P3 to promote the association of RAB5+ endosomes with mitochondria and provide insight into yet another mechanism of G1P3-induced cancer cell survival.© 2023 International Federation of Cell Biology.