Autophagy is a lysosomal degradation pathway that degrades cytosolic constituents, including whole organelles and intracellular pathogens. Previous studies on various autophagy related genes revealed the importance of the Atg12-Atg5-Atg16 complex in autophagy. Atgl6L1 is an effector of Golgi-resident Rab33B and the molecular mechanism of the interaction of Rab33B with either Atgl6L1 or in complex with Atg5 is still elusive. In the current study, using the pull down and calorimetric approaches, we have dissected the molecular insights into the interaction of Rab33B with different regions of mouse Atgl6L1 as well as with the dimeric complex, Atg5-mAtgl6L1. Our in vitro observation suggests that Atg5 is prerequisite for the augmented nucleotide dependent interaction of Rab33B with the dimeric complex, Atg5-Atgl6L1. Moreover, the results reported here suggest that Arg-24 of Atgl6L1 is crucial for its interaction with Atg5 which will have further implication in the binding of the dimeric complex to Rab33B. (C) 2016 Elsevier Inc. All rights reserved.