Rab2 in Drosophila fat and RAB2A in human cells are required for autophagosome clearance. (A and B) Tandem mCherry-GFP-Atg8a shows that autophagic flux proceeds normally in starved control cells, based on quenching of GFP (A). GFP remains fluorescent and colocalizes with mCherry in Rab2 RNAi cells (B). (C–E) Most 3xmCherry-Atg8a autophagic structures contain the lysosomal hydrolase CathL in starved control fat cells (C), whereas their overlap is reduced in rab2 mutants (D), quantified in E, n = 30–45 cells, indicating that the convergence of autophagic and lysosomal compartments is blocked in rab2 mutants. White arrows, overlapping signal; yellow arrows, mCherry-Atg8a–only vesicles in C and D. (F and G) Rab2 knockdown in GFP⁺ cells reduces the number and size of CathL vesicles (F), quantified in G, n = 9 cells. (H and I) Ultrastructural analysis of starved fat cells. Degrading autolysosomes (arrowhead) form in control cells (H), unlike in rab2 mutants (I) that accumulate double-membrane autophagosomes (asterisks) containing nondegraded cytoplasm and dense structures likely representing amphisomes (arrow). (J) Western blots reveal accumulation of p62 and both forms of Atg8a in starved rab2-null mutants, and similar protein levels in control and Rab2^GTP-expressing larvae. (K and L) Confocal analysis of human MDA-MB-231 cells transfected with scramble oligo or siRNA against RAB2A, RAB2B, or both (K). Bottom, magnification of boxed areas. Knockdown of RAB2A, but not RAB2B, causes accumulation of endogenous LC3⁺ autophagic structures, which colocalize with the late endosomal-lysosomal marker Lamp1. Quantification of LC3⁺ Lamp1⁻ autophagosomes and LC3⁺ Lamp1⁺ amphisomes (L), n = 13–23 cells. Error bars mark ± SEM in E, G, and L. Indicated channels are shown in grayscale in A–D and F.