ATG8s are essential for autophagy as they recruit various machinery to autophagic structures. We previously reported that the intracellular Ca channel TRPML3 specifically interacts with the mammalian ATG8 homolog GATE16, but not LC3B to increase autophagy. However, the underlying mechanism and the role of this specific interaction remain unclear. Here, we report that single amino acid motifs in GATE16 and TRPML3 determine the specificity of this interaction and its function in autophagy. We also discovered that RAB33B, a Golgi-resident small GTPase, functionally interacts with TRPML3 in autophagy and contains an LC3-interacting region (LIR) motif. Surprisingly, RAB33B specifically interacted with GATE16, but not with other ATG8s through an LIR motif, and disrupting this LIR motif inhibited autophagy. Upon induction of autophagy, RAB33B was recruited from the Golgi to the phagophore in an LIR-dependent manner, enhancing the interaction between RAB33B and TRPML3 while promoting autophagy. These results suggest that specific interactions involving GATE16 play a crucial role in autophagy by recruiting TRPML3 and RAB33B, forming protein complexes at the phagophore to promote autophagosome formation.