The intracellular Ca2+ channel TRPML3 regulates autophagy by multimerization in a phosphoinositide (PIP)-dependent manner; however, its role in other membrane dynamics remains unclear. Here, we report that TRPML3 regulates type III unconventional protein secretion (UcPS) through two distinct pathways: TMED10-channeled UcPS and secretory autophagy. TRPML3 interacted with TMED10 and macrophage migration inhibitory factor (MIF), a signal sequence-lacking pleiotropic cytokine whose secretion mechanism has remained undefined. We found that cytosolic MIF is transported to membrane-enclosed compartments through TMED10-channeled UcPS. Notably, TRPML3, but not TRPML1 or TRPML1/3, promoted TMED10 oligomerization, thereby enhancing MIF secretion in a Ca2+-independent manner. Furthermore, once translocated into vesicular compartments, MIF colocalized with LC3, and its secretion was dependent on secretory autophagy. Mechanistically, we identified PI(3,4)P2 as a key regulator of secretory autophagy, with TRPML1/3 acting downstream of PI(3,4)P2 to mediate Ca2+-dependent MIF secretion. Taken together, our findings demonstrate that MIF is a novel cargo of type III UcPS, operating through TMED10-channeled UcPS and secretory autophagy, which are regulated by TRPML3 in a Ca2+-independent manner and by the PI(3,4)P2-TRPML1/3 axis in a Ca2+-dependent manner, respectively.
