Selectivity and dynamics of VAP family tether exchange across membrane contact sites

To integrate diverse cellular function and coordinate homeostasis, the endoplasmic reticulum (ER) communicates with all other intracellular compartments through physical interfaces termed membrane contact sites (MCSs). Given the sheer diversity of possible MCS pairings, how the ER discriminates between simultaneously available binding partners remains unclear. To explore this, we performed comparative endogenous contact site mapping for three closely related VAP family tethers and uncovered a unique set of FFAT motif selectivity and MCS footprints for each: VAPA at mitochondria and TGN, VAPB at mitochondria and peroxisomes, and MOSPD2 at mature endosomes, lysosomes and lipid droplets. In line with their MCS selectivity profiles, co-depletion of VAPA and VAPB, but not MOSPD2, caused systemic breakdown in mitochondrial integrity, while silencing of MOSPD2 alone was sufficient to disrupt the organization and transport of (endo)lysosomes. Unexpectedly, coincident loss of VAPA and VAPB instigated ER contact site rewiring by redirecting endogenous MOSPD2 to the collapsing mitochondrial network. Our findings define hierarchies of ER contact site formation and reveal compensation mechanisms exploited by cells to safeguard organelle homeostasis and crosstalk.