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. 2021 Feb 17;118(8):e2014941118. doi: 10.1073/pnas.2014941118

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Fig. 4. — Proline hinges and specific aromatic residues predicted to face the inward-facing cavity of PQLC2 are required for WDR41 binding. (A) Structural representation of PQLC2 transporter (front view), showing the side chains of prolines 55 and 201 (magenta) and aromatic residues predicted to line the inward-facing cavity (F49, W78, Y105, and Y195; yellow). (B) HeLa cells transiently cotransfected with WDR41-HA and the indicated PQLC2-FLAG mutants were lysed and immunoprecipitated (anti-FLAG M2 affinity gel) and immunoblotted with HA and FLAG antibodies. (C) HeLa cells transiently cotransfected with plasmids expressing GFP-TIP and the indicated PQLC2 mutants were lysed and immunoprecipitated (IP: GFP) and immunoblotted with FLAG antibody. Experiments in B and C were independently replicated at least three times. (D) Spinning-disk confocal immunofluorescence microscopy of transiently transfected HeLa cells expressing PQLC2-FLAG with the indicated mutations along with detection of endogenous LAMP1 (late endosomes and lysosomes). (Insets) 3× magnifications of the selected regions denoted by dashed lines. (Scale bars: 10 µm.)