KEY RESOURCES TABLE
| Reagent or Resource | Source | Identifier |
|---|---|---|
| Antibodies | ||
| Mouse anti-human LAMP1 (H4A3) antibody | DSHB | Cat# h4a3, RRID: AB_2296838 |
| Rat anti-HA (3F10) antibody | Roche | Cat# 11867423001, RRID: AB_390918 |
| Rabbit Anti-Human alpha Synuclein, phospho (Ser129) antibody | Abcam | Cat# ab51253, RRID: AB_869973 |
| Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Texas Red-X | Thermo Fisher Scientific | Cat# T-862, RRID: AB_2556781 |
| Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | Cat# A-11006, RRID: AB_2534074 |
| Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | Cat# A-21202, RRID: AB_141607 |
| Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | Cat# A-21206, RRID: AB_253579 |
| Mouse anti-GFP (clone 7.1 and 13.1) antibody | Roche | Cat# 11814460001, RRID: AB_390913 |
| Rabbit anti-Phospho-Akt(Thr308) (C31E5E) antibody | Cell Signaling Technology | Cat #2965, RRID: AB_2255933 |
| Rabbit anti-Akt (pan) (C67E7) antibody | Cell Signaling Technology | Cat# 4691, RRID: AB_915783 |
| Rabbit anti-Cathepsin B antibody [EPR4323] | Abcam | Cat# ab125067, RRID: AB_10972167 |
| Rabbit anti-Cathepsin D antibody [EPR3057Y] | Abcam | Cat# ab75852, RRID: AB_1523267 |
| Mouse Anti-beta-Actin Antibody (AC-74) | Sigma-Aldrich | Cat# A5316, RRID: AB_476743 |
| IRDye® 680RD Goat anti-mouse IgG (H+L) secondary antibody | Li-cor | Cat# 926-68070, RRID: AB_10956588 |
| IRDye® 800CW Goat anti-Rabbit IgG (H+L) secondary antibody | Li-cor | Cat# 926-32211, RRID: AB_621843 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| N-methyl-D-glucamine (NMDG) | Sigma-Aldrich | M2004; CAS: 6284-40-8 |
| Methanesulfonic acid (MSA) | Sigma-Aldrich | 471356; CAS: 75-75-2 |
| HCl | Fisher Scientific | A144C-212; CAS: 7647-01-0 |
| Tetramethylammonium (TMA) | Sigma-Aldrich | T-7505; CAS: 10424-65-4 |
| K-Gluconate | Sigma-Aldrich | P1847; CAS: 299-27-4 |
| KOH | Fisher Scientific | P250-500; CAS: 1310-58-3 |
| NaOH | Fisher Scientific | S318-500; CAS: 1310-73-2 |
| KCl | Sigma-Aldrich | P9541; CAS: 7447-40-7 |
| NaCl | Sigma-Aldrich | S9888; CAS: 7647-14-5 |
| CaCl2 | Sigma-Aldrich | C8106; CAS: 10035-04-8 |
| MgCl2 | Sigma-Aldrich | 208337; CAS: 7786-30-3 |
| NH4Cl | Sigma-Aldrich | A4514; CAS: 12125-02-9 |
| NaH2PO4 | Sigma-Aldrich | S3139; CAS: 7558-80-7 |
| Sucrose | Sigma-Aldrich | S0389; CAS: 57-50-1 |
| Tetraethylammonium chloride (TEA-Cl) | Sigma-Aldrich | T2265; CAS: 56-34-8 |
| Cs-Methanesulfonate (Cs-MSA) | Sigma-Aldrich | C1426; CAS: 2550-61-0 |
| HEPES | Sigma-Aldrich | H3375; CAS: 7365-45-9 |
| MES | Sigma-Aldrich | M8250; CAS: 1266615-59-1 |
| Citric acid | Sigma-Aldrich | 27109; CAS: 77-92-9 |
| Acetic acid | Fisher Scientific | A38-212; CAS: 64-19-7 |
| Ethanol | Decon laboratories inc. | 2701; CAS: 64-17-5 |
| Methanol | Fisher Scientific | A52-4; CAS: 67-56-1 |
| Isopropanol | Fisher Scientific | A416-4; CAS: 67-63-0 |
| Glucose | Sigma-Aldrich | G6152; CAS: 50-99-7 |
| Triton-X 100 | Sigma-Aldrich | T8787; CAS: 9002-93-1 |
| NaF | Sigma-Aldrich | S7920; CAS: 7681-49-4 |
| Na-orthovanadate | Sigma-Aldrich | S6508; CAS: 13721-39-6 |
| EDTA | Sigma-Aldrich | 03609; CAS: 60-00-4 |
| EGTA | Sigma-Aldrich | E4378; CAS: 67-42-5 |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2650; CAS: 67-68-5 |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A3059; CAS: 9048-46-8 |
| Paraformaldehyde (PFA) 4% in PBS | Thermo Fisher Scientific | J19943-K2 |
| Phosphate-buffered saline (PBS) | Gibco | 10010 |
| RIPA | Boston Bioproducts | BP-115 |
| Protease inhibitor cocktail | Sigma Aldrich | P8340 |
| Phosphatase inhibitor cocktail I | Sigma Aldrich | P0044 |
| LDS sample buffer (4x) | Invitrogen | NP0007 |
| Pierce™ BCA™ Protein Assay | Invitrogen | 23227 |
| PVDF membrane | Millipore | IPFL00010 |
| Fluoromount-G | Southern Biotech | 0100-01 |
| Magic Red Cathepsin B assay kit | ImmunoChemistry Technologies | 937 |
| Pepstatin A BODIPY™ FL | Invitrogen | P12271 |
| DQ™-BSA red dye | Invitrogen | D12051 |
| CF®488a conjugated dextran-10 kd | Biotium | 80110 |
| CF®555 conjugated dextran-10 kd | Biotium | 80112 |
| LysoSensor™ Yellow/Blue DND-160 | Invitrogen | L7545 |
| LysoTracker™ Red DND-99 | Invitrogen | L7528 |
| pHrodo™ Green conjugated dextran-10 kd | Invitrogen | P35368 |
| Oregon Green™ 488 conjugated dextran-10 kd | Invitrogen | D7170 |
| pHrodo™ Red AM | Invitrogen | P35372 |
| MitoTracker™ Green FM | Invitrogen | M7514 |
| 4′,6-diamidino-2-phenylindole (DAPI) | Sigma Aldrich | D9542; CAS: 28718-90-3 |
| Lipofectamine 2000 | Invitrogen | 11668-027 |
| DMEM medium without phenol red | Gibco | 21063029 |
| DMEM medium high glucose | Gibco | 11965 |
| Iscove’s Modified Dulbecco’s Medium (IMDM) | Gibco | 12440 |
| Neurobasal™ Medium | Gibco | 21103049 |
| Fetal bovine serum (FBS) | Gemini Bio-Products | 100-106 |
| B-27™ Supplement (50X), serum free | Gibco | 17504044 |
| Trypsin-EDTA (0.05%), phenol red | Gibco | 25300054 |
| Puromycin Dihydrochloride | Gibco | A1113803 |
| Antibiotic-Antimycotic | Gibco | 15240062 |
| MycoAlertTM Mycoplasma Detection Kit | Lonza | LT07-218 |
| Poly-D-lysine solution | Millipore | A-003-E |
| Poly-L-lysine hydrobromide | Sigma Aldrich | P1399; CAS: 25988-63-0 |
| Vacuolin-1 | Sigma Aldrich | 673000; CAS: 351986-85-1 |
| 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) | Tocris Bioscience | 1540; CAS: 82749-70-0 |
| 2,7-Dichloro-9,10-dihydro-9,9-dimethyl-10-[2-(2H-tetrazol-5-yl)ethyl]acridine (ML 67-33) | Tocris Bioscience | 6886; CAS: 1443290-89-8 |
| Bafilomycin-A1 | Cayman Chemical | 11038; CAS: 88899-55-2 |
| Arachidonic acid-sodium | Cayman Chemical | 10006607; CAS: 6610-25-9 |
| Arachidonic acid | Cayman Chemical | 90010; CAS: 506-32-1 |
| SC-79 | Cayman Chemical | 14972; CAS: 305834-79-1 |
| MK-2206 | Cayman Chemical | 11593; CAS: 1032350-13-2 |
| ARQ-092 | Cayman Chemical | 21388; CAS: 1313881-70-7 |
| Wortmannin | Cayman Chemical | 10010591; CAS: 19545-26-7 |
| Latrunculin B from Latruncula magnifica | Sigma Aldrich | L5288; CAS: 76343-94-7 |
| PI(3,5)P2 diC8 | Echelon Biosciences | P-3508 |
| ML-SA5 | This paper | N. A. |
| isoflurane (Fluriso™) | VetOne | 501017 |
| Tissue-Tek® O.C.T. (Optimal Cutting Temperature) compound | Sakura Finetek | 4583 |
| Recombinant mouse WT α-synuclein preformed fibril (5 mg/mL) | a gift from Dr. Kelvin C. Luk, the University of Pennsylvania Perelman School of Medicine | N. A. |
| Critical Commercial Assays | ||
| QuikChange II Site-Directed Mutagenesis Kit | Agilent | 200523 |
| Gibson Assembly Cloning Kit | New England Biolabs | E5510S |
| Q5® High-Fidelity DNA Polymerase | New England Biolabs | M0491S |
| GoTaq™ master mixes | Promega | M7122 |
| Experimental Models: Cell Lines | ||
| Monkey: COS1 | ATCC | CRL-1650, RRID: CVCL_0223 |
| Human: HEK293T cells | ATCC | CRL-3216; RRID: CVCL_0063 |
| Human: HeLa cells | ATCC | CCL-2; RRID: CVCL_0030 |
| Human: HAP1 cells | Horizon Discovery Ltd. | C631; RRID: CVCL_Y019 |
| Human: TMEM175 KO HAP1 cells | This paper | N. A. |
| Human: TMEM175-HA Knock-in HAP1 cells | This paper | N. A. |
| Human: TPC1&TPC2 DKO HAP1 cells | This paper | N. A. |
| Human: LRRC8A KO HAP1 cells | (Li et al., 2020b) | N. A. |
| Human: TMEM175 KO HEK293T cells | This paper | N. A. |
| Human: HEK293T cells stably overexpressing human TMEM175 (L1&L2) | This paper | N. A. |
| Human: TMEM175 KO HeLa cells (KO-1&KO-2) | This paper | N. A. |
| Human: TMEM175 KO HeLa cells stably re-expressing human TMEM175 | This paper | N. A. |
| Human: TMEM175 KO HeLa cells stably re-expressing human TMEM175-D41A | This paper | N. A. |
| Human: TRPML1-KO fibroblasts | Coriell Institute for Medical Research | GM02048; RRID: CVCL_M927 |
| Experimental Models: Organisms/Strains | ||
| Mouse: Tmem175 −/− / C57BL/6N | This paper | N. A. |
| Oligonucleotides | ||
| sgRNA targeting sequence: hTMEM175 (#1): 5’- CGCGTGGAAGCCTTCAGCGA -3’ | http://crispr.mit.edu | N. A. |
| sgRNA targeting sequence: hTMEM175 (#2): 5’- TGTCCTCCCTGGTCCATCTC -3’ | http://crispr.mit.edu | N. A. |
| sgRNA targeting sequence: hTMEM175 (#3): 5’- CGGCCGCCAAACCACACCGA -3’ | http://crispr.mit.edu | N. A. |
| sgRNA targeting sequence: mTmem175 (#1): 5’- GCACTAATACTCTTAGCCTG -3’ | http://crispr.mit.edu | N. A. |
| sgRNA targeting sequence: mTmem175 (#2): 5’- GGACCCCTTACAATATGCCT -3’ | http://crispr.mit.edu | N. A. |
| Genotyping primer: 5’- CATTCCTGTTGCTCCGTCTCCTTGT -3’ and 5’- CGCCAGGGAAGCTCTGTACTTTTGA -3’ | This paper | N. A. |
| for Tmem175−/− mice | ||
| Genotyping primer: 5’- TGCTATGCTGAGCTTATAGCCTTG -3’ and 5’- AACGTTCCAAGAAGGAGGATCGCC -3’ for Tmem175−/− mice | This paper | N. A. |
| Recombinant DNA | ||
| pEZ-M55-mCherry-hTMEM175 | GeneCopoeia | EX-V0772-M55 |
| pEZ-M29-EGFP-hTMEM175 | GeneCopoeia | EX-V0772-M29 |
| pEZ-M97-hOTOP1-EGFP | GeneCopoeia | EX-E1341-M97 |
| pSpCas9 (BB)-2A-puro vector | Addgene | 48139 |
| pHluorin (in modified pEYFP-N1) | This paper | N. A. |
| Software and Algorithms | ||
| MetaMorph 7.10 | Molecular Devices | https://www.moleculardevices.com/ |
| pClamp 10.7 | Molecular Devices | https://www.moleculardevices.com/ |
| OriginPro 2018 | Originlab | https://www.originlab.com/ |
| GraphPad Prism 8 | Graphpad | https://www.graphpad.com/ |
| Fiji/ImageJ | Fiji contributors | https://imagej.net/Fiji |
| Image Studio lite | Li-cor | https://www.licor.com/ |