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. 2022 Feb 23;603(7899):159–165. doi: 10.1038/s41586-022-04431-8

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 7 — a, PEN2 interacts with ATP6AP1. HEK293T cells were transfected with HA-tagged PEN2 and Myc-tagged ATP6AP1 (AP1). Cells were lysed, and 10 μM metformin (final concentration) or PBS was added to the lysates. Immunoprecipitation (IP) was performed using antibodies against HA, followed by immunoblotting with antibodies indicated. b, PEN2 interacts with ATP6AP1 in vitro. Some 1 μg of FLAG-tagged PEN2 (expressed in HEK293T cells, and purified through eluting with FLAG® peptide) were incubated with 1 μg of Strep-tagged ATP6AP1 (expressed in HEK293T cells, and purified through eluting with desthiobiotin) (input) in lysis buffer, then with metformin at indicated concentrations for 2 h. Immunoprecipitation was performed using ANTI-FLAG® M2 Affinity Gel, followed by immunoblotting with antibodies indicated. c, Domain mapping for the region on ATP6AP1 responsible for PEN2-binding. HA-tagged PEN2 was co-transfected with Myc-tagged ATP6AP1, or its deletion mutants into HEK293T cells. Immunoprecipitation was performed using antibody against Myc-tag, followed by immunoblotting with antibodies indicated. d, Replacement of ATP6AP1 transmembrane domain with that of LAMP2, blocks its interaction with PEN2. HEK293T cells transfected with HA-tagged PEN2-D90A, along with Myc-tagged LAMP2^TM-ATP6AP1 or wildtype ATP6AP1, were lysed, and 10 μM metformin was added to the lysates, followed by immunoprecipitation with antibody against HA, and immunoblotting with antibodies indicated. e, In silico modelling of ATP6AP1 (cyan) bound to PEN2 (magenta). Circled area indicates the predicted interface, in which residues 27, 28, 30, 31, 34, 38, 42, 43, 57, 58, 60, 63 to 65, 67, 68, 71, 72, 74 and 75, within the transmembrane domain of PEN2, are involved. f, ATP6AP1 shows much weaker interaction with other γ-secretase subunits than PEN2. MEFs were lysed and incubated with metformin as in a, followed by immunoprecipitation with antibodies against ATP6AP1, or PEN2 as a control. Immunoprecipitants were than subjected to immunoblotting with antibodies against PS1, PS2, NCSTN, APH1A/B/C, as well as PEN2 and ATP6AP1. g, Metformin, through promoting the association between PEN2 and ATP6AP1, enhances association of ATP6AP1 and γ-secretase. PEN2^-/- MEFs infected with lentivirus expressing HA-tagged PEN2 or its 20A mutant (lacking the interface for ATP6AP1) were lysed and incubated with metformin as in a, followed by immunoprecipitation with antibodies against ATP6AP1. Immunoprecipitants were than subjected to immunoblotting with antibodies against PS1, PS2, NCSTN, APH1A/B/C, as well as PEN2 and ATP6AP1. h, ATP6AP1 does not interact with metformin. HEK293T cells transfected with HA-tagged ATP6AP1, or HA-tagged PEN2 as a control, were lysed, followed by analysing the interaction between ATP6AP1 or PEN2 with Met-P1 as in Extended Data Fig. 2d. i, Strategies to generate MEFs (lower panel) or HEK293T cells (upper panel) with knockout of ATP6AP1. sgRNAs against ATP6AP1, whose sequences are listed in Methods section, were applied to generate ATP6AP1^-/- MEFs and HEK293T cells. j, Knockout of ATP6AP1 leads to constitutive activation of AMPK. MEFs with ATP6AP1 knocked out, along with its wildtype control, were incubated with metformin at indicated concentrations for 12 h, followed by analysing p-AMPK and p-ACC. k, Knockout of ATP6AP1 renders v-ATPase inactive. ATP6AP1^-/- MEFs were treated with 200 μM metformin for 12 h, followed by analysis of lysosomal pH with the Lysosensor dye. Data (relative intensity of Lysosensor, processed as in Fig. 1a) were graphed as mean ± s.e.m., n = 29 (control) and 26 (metformin-treated) cells from 6 dishes/experiment for WT MEFs, and 28 (control) and 23 (metformin-treated) cells from 4 dishes/experiments for ATP6AP1^-/- MEFs, P value within each genotype was determined by two-sided Mann-Whitney test (for WT MEFs), or by two-sided Student’s t-test (for ATP6AP1^-/- MEFs). l, Validation data showing that the re-introduced ATP6AP1 and its mutants are expressed at a close-to-endogenous level in ATP6AP1^-/- MEFs (upper panel) and HEK293T cells (lower panel). Experiments in this figure were performed three times, except b, f, h, four times and l five times.

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