Proteolytic control of mitochondrial calcium transport by intermembrane-space proteases

The mitochondrial intermembrane space (IMS) is a critical regulatory interface for mitochondrial calcium ( _m Ca ²⁺ ) flux. Positioned between the outer and inner mitochondrial membranes, the IMS links cytosolic Ca ²⁺ signal to regulated Ca ²⁺ uptake into the matrix. This positioning allows the IMS to influence _m Ca ²⁺ transport and Ca ²⁺ -dependent mitochondrial metabolism. _m Ca ²⁺ homeostasis is governed mainly by the mitochondrial calcium uniporter complex (mtCU), which mediates _m Ca ²⁺ uptake, and the Na ⁺ /Ca ²⁺ exchanger NCLX, which drives _m Ca ²⁺ efflux. However, whether IMS regulatory events, particularly proteolytic remodeling by IMS proteases, control this transport machinery remains unclear. Using complementary knockout and overexpression approaches targeting ten IMS proteases (NLN, ATP23, IMMP1L, IMMP2L, YME1L1, OMA1, LACTB, LACTB2, PARL, and HTRA2), we identified protease-specific remodeling of mtCU components and NCLX abundance. Transcriptomic and proteomic analyses showed that these changes arise largely from protease-specific control of transporter stability rather than transcriptional regulation alone. Proximity-labeling proteomics further revealed spatial associations between IMS proteases and _m Ca ²⁺ transport components. Functionally, perturbing IMS proteases altered _m Ca ²⁺ flux and reduced _m Ca ²⁺ retention capacity, indicating impaired buffering against Ca ²⁺ overload. Together, these findings identify IMS proteases as a proteostatic regulatory network controlling _m Ca ²⁺ transport and establish a mechanistic link between mitochondrial proteostasis and Ca ²⁺ homeostasis.