Membrane cholesterol modulates STEAP2 conformation during dynamic intracellular trafficking processes leading to broad subcellular distribution

STEAP2 is a member of the Six-Transmembrane Epithelial Antigen of the Prostate (STEAP) protein family that is proposed to function as metalloreductase. While STEAP2 shows a complex subcellular distribution pattern localizing to both secretory and endocytic pathway organelles, how such broad steady-state distribution is maintained is unknown. Similarly, whether STEAP2 undergoes any compartment-specific modulation during intracellular trafficking has not been reported. Leveraging a newly-identified monoclonal antibody that recognizes a conformation-sensitive epitope nested in the second extracellular loop of STEAP2, we demonstrate that the epitope formation was dependent on the cholesterol content of the membrane in which STEAP2 was embedded. Monitoring the STEAP2-dependent internalization of this antibody uncovered STEAP2's rapid internalization from the cell surface and their subsequence trafficking to the Golgi region and endosome-like puncta. Acute inhibition of endocytosis also increased the detectable amount of STEAP2 at the plasma membrane. Collectively, these experiments demonstrate that an intricate balance of membrane flux between the secretory and endocytic pathways underlies the characteristic broad subcellular localization of STEAP2. By using a cell-based assay that detects the metalloreductase functions of cell surface-localizing STEAP4, STEAP2's metalloreductase activities were not detectable, suggesting that its enzymatic function is suppressed at the plasma membrane. The conformational modulation of STEAP2 by the local membrane cholesterol content can therefore serve as a potential mechanism to modulate STEAP2 function in a compartment-restricted manner, by coupling a pre-existing difference in cholesterol content among different cellular membranes to a dynamic trafficking process leading to broad subcellular distribution. •STEAP2 distributes broadly into multiple subcellular compartments at steady state.•Cell surface STEAP2 is a minor constituent of the total cellular STEAP2 protein pool.•STEAP2 conformation is modulated within the membrane by its cholesterol content.•STEAP2's metalloreductase activities are suppressed at the plasma membrane.•STEAP2 properties are controlled in a localization-dependent manner by cholesterol.