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Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner.

The small GTPase Rab34 regulates spatial distribution of the lysosomes, secretion, and macropinocytosis. In this study, we found that Rab34 is over-expressed in aggressive breast cancer cells, implying a potential role of Rab34 in breast cancer. Silencing Rab34 by shRNA inhibits cell migration, invasion, and adhesion of breast cancer cells. Rab34 specifically binds to the cytoplasmic tail of integrin β3, and depletion of Rab34 promotes the degradation of integrin β3. Interestingly, EGF induces the translocation of Rab34 to the membrane ruffle, which is greatly enhanced by the expression of Src kinase. Accordingly, Rab34 is tyrosine phosphorylated by Src at Y247 residue. A mutant mimicking phosphorylated form of Rab34 (Rab34Y247D) promotes cell migration and invasion. Importantly, the tyrosine phosphorylation of Rab34 is inhibited in cells in suspension, and increased with the cells re-adhesion. In addition, Rab34Y247D promotes cell adhesion, and enhances integrin β3 endocytosis and recycling. The results uncover a role of Rab34 in migration and invasion of breast cancer cells and its involvement in cancer metastasis, and provide a novel mechanism of tyrosine phosphorylation of Rab34 in regulating cell migration, invasion, and adhesion through modulating the endocytosis, stability, and recycling of integrin β3.