Pmturquoise2 peroxi

These plasmids were a gift from Dorus Gadella: pmTurquoise2-Mito (Addgene plasmid #36208), pmTurquoise2-ER (Addgene plasmid #36204), pmTurquoise2-Peroxi (Addgene plasmid #36203), pmTurquoise2-Golgi (Addgene plasmid #36205) and pmTurquoise2-H2A (Addgene plasmid #36207)^{59 (link)}. pcDNA3.1-MCS-BirA-R118G-HA was a gift from Kyle Roux (Addgene plasmid # 36047)^{55 (link)}. RFP-Dcp1a and RFP-TIA1 were described before^{60 (link)}. MACROD1, MACROD2 and OARD1 (encoding TARG1) were amplified from Hela cDNA using primers with flanking attB sites suitable for the Gateway System (Invitrogen) and inserted into pDONR/zeo with a Gateway BP reaction. pcDNA5/FRT/TO-MACROD1, -MACROD2 or -TARG1 were generated by performing a Gateway LR reaction according to the manufacturer’s instructions. GFP-labelled plasmids were generated by LR reactions into pDEST47 and pDEST53. pcDNA3-mRuby2-MACROD1 and pcDNA3-mRuby2-MACROD1d77 were generated using primers with flanking HindIII and Nhel restriction sites for subsequent insertion into pcDNA3-mRuby2 for which the pcDNA5 constructs were used as PCR template. pcDNA3-BirA-R118G constructs with full length MACROD1, MACROD2 and TARG1 were generated using primers with flanking EcoRI and BamHI restriction sites and the pcDNA5 constructs as template.

The expression plasmids pEGFP-hGalectin-3 (GFP-Gal3) and pmRFP-EGFP-Galectin-3 (ptf-Gal3) were purchased from Addgene [deposited by Dr. Tamotsu Yoshimori (Osaka University, Japan)]. The subcellular localization vectors, pmTurquoise2-ER, pmTurquoise2-Golgi, and pmTurquoise2-Peroxi, in which a cyan fluorescence protein (Turquoise) was fused with ER, Golgi, or peroxisome targeting sequences respectively, were obtained from Addgene [deposited by Dr. Dorus Gadella (University of Amsterdam, Netherlands)]. pEGFP-TFEB was obtained from Addgene [deposited by Dr. Shawn Ferguson (Yale Univeristy, USA)]. pLAMP1-GFP and pEGFP-LC3 were kindly provided by Dr. Peter K. Kim (Toronto University, Canada) and Dr. Noboru Mizhushima (Tokyo University, Japan), respectively. pCMV-lyso-pHluorin (Lyso-pHluorin) was purchased from Addgene [deposited by Dr. Christian Rosenmund (Neuroscience Research Center, Germany)]. pEGFP-Ubiquitin (GFP-Ub) and pLAMP1-mCherry were purchased from Addgene [deposited by Dr. Nico Dantuma (Karolinska Institutet, Sweden) and Amy Pamer (University of Colorado, USA)], respectively. Mitochondrial-YFP (pMito-YFP) plasmid was kindly provided by Dr. Gyesoon Yoon (Ajou University, Korea).

BEAS-2B, A549, HeLa–Parkin, and MEF cells were maintained in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum (FBS; JR Scientific Inc., Woodland, CA, USA). PINK1 KO MEFs were kindly provided by Dr. Jongkyeong Chung (Seoul National University, Seoul, Republic of Korea) [50 (link)]. Cell lines stably expressing mt-Keima or Keima were generated via infection with a lentivirus produced by using a pLVX-mtKeima lentiviral construct [23 (link)].
pLV-ER GFP (#80069), pLV-Golgi eGFP (#79809), and pmTurquoise2-Peroxi (#36203) were obtained from Addgene (Watertown, MA, USA). A mitochondrial YFP-expressing plasmid (pLESIP-mitoYFP) was generated by subcloning mitoYFP from pcDNA3-mitoYFP (provided by Dr. Gyesoon Yun, Ajou University, Suwon, Republic of Korea) into the pLESIP vector. CCCP (C2759), berberine (14050), bafilomycin A1 (B1793), and compound C (171260) were purchased from Sigma-Aldrich (St. Louis, MO, USA).