1. Unconventional Trafficking of Mammalian Phospholipase D3 to Lysosomes
Adriana Carolina Gonzalez, Michaela Schweizer, Sebastian Jagdmann, Christian Bernreuther, Thomas Reinheckel, Paul Saftig, Markus Damme 
Cell Reports 
Volume 22, Issue 4, Pages (January 2018)
DOI: /j.celrep
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2. Cell Reports 2018 22, 1040-1053DOI: (10.1016/j.celrep.2017.12.100)
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3. Figure 1 PLD3 Localizes to Late Endosomes and Lysosomes
(A) Schematic representation of the epitope-binding sites of the PLD3-specific antibodies. hPLD3, human PLD3; mPLD3, mouse PLD3.
(B) Western blot analysis of human and murine PLD3 cDNA transfected HeLa or Neuro2a cells (left panel) and endogenous PLD3 in wild-type (WT) and PLD3 KO HeLa cells (right panel) followed by detection with the N-terminal- and the luminal-domain-specific antibodies. ∗ indicates nonspecific signal. fl, full length; nt, N terminus.
(C) Co-immunofluorescence of HeLa cells transfected with hPLD3 and stained for various endogenous organelle markers (red; KDEL, GM130, EEA1, LBPA, and LAMP2) and antibodies against the N terminus (amino acids 1–20; left row, green) or the luminal part of PLD3 (amino acids 93–218; right row, green). Nuclei are stained with DAPI (blue). Pearson correlation coefficient with both antibodies with the respective organelle marker is depicted in the graphs. Error bars represent SEM. ∗∗∗p < 0.001; unpaired Student’s t test. The scale bars represent 10 μm. n = 12.
(D) In SH-SY5Y neuroblastoma cells differentiated for 4 days with retinoic acid, the luminal domain of endogenous PLD3 (red) co-localizes with the lysosomal marker LAMP2 (green). The PLD3 signal disappears in cells transfected with a GFP-containing PLD3 CRISPR/Cas9 plasmid (∗).
(E) Immunofluorescence staining on non-neurological human brain sections (cerebral cortex) shows vesicular staining pattern with co-localization of PLD3 (green) and LAMP2 (red). The scale bars represent 10 μm. Pearson correlation coefficient is depicted; n = 12.
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4. Figure 2
PLD3 Is Proteolytically Processed, Yielding a Stable Luminal Soluble Tail
(A) Western blot analysis of PLD3-transfected HeLa cells (left panel) and mouse brain lysates (right panel) after treatment with the glycosidases EndoH or PNGaseF followed by detection with the PLD3-specific antibodies. β-glucocerebrosidase (β-GC), known to be modified with both complex- and high-mannose-type glycans, is shown as a positive control for glycosidase treatment. ∗ indicates bands derived from previous incubation with the luminal antibody and inefficient stripping.
(B) Kinetic analysis of detection of PLD3 polypeptides at different time points after transfection.
(C) Immunoblots of PLD3-transfected HeLa cells with our without β-mercaptoethanol (β-ME).
(D) Membrane separation of PLD3-transfected HeLa cells after cell lysis into a soluble protein and membrane protein fraction by ultracentrifugation.
(E) Confocal microscopy of HeLa cells transfected with PLD3 and Myc-tagged Rab5-WT (upper panel) or the dominant active form of Rab5 (Q79L; lower panel, red), followed by staining with the PLD3-specific antibodies (green). Nuclei are stained with DAPI (blue). Inset 1: membranous staining with the N-terminal antibody is observed in most vesicles. Inset 2: additional intraluminal dot-like staining is observed in some vesicles. Profile plots are depicted for the indicated antibodies and vesicles labeled with a white line.
(F) Co-staining with the antibodies against the PLD3 N terminus (left) or luminal domain (right, green), respectively, with LAMP2 (red) after transfection with Rab5-Q79L.
(G) Co-staining of PLD3 (green) with CD63 (red) shows high degree of co-localization N-terminal antibody against PLD3 (left panel) in intraluminal vesicles and with the luminal PLD3 antibody (right panel). The scale bars represent 10 μm.
(H) Fractionation of organelles from PLD3-transfected HeLa cells by Percoll density gradient centrifugation shows a clear separation of the full-length protein and the luminal domain of PLD3. The relative distribution of the lysosomal marker enzyme β-hexosaminidase activity is depicted below.
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5. Figure 3
Proteolytic Processing of PLD3 Is Mediated by Acidic Cysteine Proteases
(A–C) Western blot analysis with antibodies against the N terminus and the luminal domain of PLD3-transfected HeLa cells after treatment with the indicated inhibitors for intracellular transport (A) monensin and brefeldin A; (B) inhibitors interfering with lysosomal pH, including NH4Cl, bafilomycin A, and chloroquine; and (C) the protease inhibitors E64D, leupeptin, and pepstatin A. GAPDH is shown as loading control. The ratio between the full-length protein and the luminal domain after densitometric quantification is shown. Error bars represent SEM. ∗∗∗p < 0.001; ∗∗p < 0.01; unpaired Student’s t test (n = 3).
(D) Co-immunofluorescence of PLD3 (green, antibody against the N terminus left panel, antibody against the luminal domain right panel) with LAMP2 (red) after treatment with different inhibitors or DMSO as control. Nuclei are stained with DAPI (blue). The scale bars represent 10 μm.
(E) Western blot analysis with antibodies against the N terminus and the luminal domain of PLD3 on mouse brain of wild-type control and CtsB/L double-deficient mice. Quantification of full-length PLD3 and the proteolytic fragments of PLD3 from two wild-type and two CtsB/L double-deficient mice. The average signal intensity for each fragment of the wild-type is set as 1. ∗ indicates nonspecific signal.
(F) Scheme of the proteolytic events leading to the liberation of the stable luminal domain and the degradation of the N-terminal fragment by CtsB and CtsL.
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6. Figure 4
PLD3 Partially Localizes to MVBs, and Transport to MVBs Depends on PtdIns(3)P or PtdIns(3,5)
(A) Double-immunogold labeling of PLD3-transfected HeLa cells with antibodies against the N terminus (I and III) and the luminal domain (II, IV, and V) of PLD3 (10-nm gold particles; white arrowheads) and LAMP1 (15-nm gold particles; black arrowheads). PLD3 is detected in intraluminal vesicles of MVBs and in the lumen of lysosomes whereas LAMP1 is only found on the limiting membrane. Occasionally, PLD3 labeling is seen at the plasma membrane. The scale bars represent 500 nm.
(B) Confocal microscopy of PLD3-transfected HeLa cells treated with 100 nM wortmannin (or DMSO alone), followed by staining with the PLD3-specific antibodies (green) and LAMP1 (red, upper panel). Profile plots for both channels are depicted for the indicated insets. As a positive control of wortmannin treatment, PLD3 (green) was stained with CD63 (red, lower panel). Nuclei are stained with DAPI (blue). The scale bars represent 10 μm.
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7. Figure 5 The Transport of PLD3 to Lysosomes Depends on ESCRT
(A) Co-immunofluorescence of PLD3- and Hrs-GFP (green) co-transfected HeLa cells (upper panel) and of the same cells with LAMP2 (green, lower panel) and antibodies against the N terminus and the luminal domain of PLD3 (red), respectively. Nuclei are stained with DAPI (blue). The scale bars represent 10 μm.
(B) Western blot analysis of PLD3 and Hrs double-transfected cells with antibodies against the N terminus and the luminal domain of PLD3, GFP, and GAPDH. The ratio between the luminal domain of PLD3 and the full-length protein is depicted.
(C) Co-immunofluorescence of PLD3- and Vps4-WT or Vps4a-E228Q co-transfected HeLa cells with LAMP2 (red) and antibodies against the N terminus and the luminal domain of PLD3 (green), respectively. The scale bars represent 10 μm. PCC of each PLD3 antibody with LAMP2 is depicted in the bar graph. Error bars represent SEM. ∗∗∗p < 0.001; ∗∗p < 0.01; unpaired Student’s t test.
(D) Western blot analysis of PLD3-transfected HeLa cells co-transfected with two different dominant-negative mutants of Vps4a (Vps4a-E228Q-3×FLAG or Vps4a-K173Q-GFP) or their corresponding Vps4a wild-type plasmids with antibodies against the N terminus and the luminal domain of PLD3. Cathepsin D is processed normally in Vps4a-transfected cells (imm., immature; int., intermediate; mat., mature). The ratio between the luminal domain of PLD3 and the full-length protein in each WT and mutant Vps4a transfected is depicted. Error bars represent SEM. ∗∗∗p < 0.001; ∗p < 0.05; unpaired Student’s t test.
(E) siRNA-mediated knockdown of Hrs in PLD3-transfected HeLa cells. Levels of Hrs are reduced to ∼20%. The ratio between the luminal domain of PLD3 and the full-length protein in scrambled- (Scr) and Hrs-siRNA transfected cells is represented in the bar graph. Error bars represent SEM. ∗∗p < 0.01; unpaired Student’s t test. GAPDH is depicted as loading control.
(F) Immunogold labeling of PLD3-transfected HeLa treated with Hrs-siRNA cells with the antibody against the luminal domain (arrowheads, 15-nm gold).
(G) Scheme summarizing the sorting and processing events for PLD3 on its route to lysosomes.
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8. Figure 6 The N Terminus of PLD3 Is Ubiquitinated
(A) Immunoprecipitation of PLD3 and HA-tagged ubiquitin co-transfected HeLa cells with antibodies against PLD3 (left panel) followed by immunoblot for HA (upper panel) and PLD3 (lower panel). Immunoprecipitation from the same cell lysates immunoprecipitated with antibodies against HA (right panel) followed by immunoblot for PLD3 (upper panel) and HA (lower panel).
(B) Alignment of the N terminus of PLD3 orthologs in different mammals. Lysine residues are highlighted in red.
(C) Ubiquitination assay using tandem ubiquitin-binding entity. HeLa cells were transfected with wild-type and mutant PLD3 with replacement of each lysine residue in the N terminus to arginine (PLD3 (K1-6R)) followed by addition of GST or GST-TUBE and analyzed by immunoblot (lysates) or affinity purification with glutathione affinity resin and subsequent immunoblot with indicated antibodies against PLD3 (upper panels) and GST (lower panel). Glutathione affinity resin alone served as a negative control. Ratio of the signal intensity between GST-pull-down and lysate is shown.
(D) Proposed scheme for the ESCRT- and ubiquitin-dependent sorting of PLD3 into ILVs. Ubiquitinated PLD3 is recognized by ESCRT-0 (Hrs) and de-ubiquitinated, and ILVs are generated by Vps4.
Cell Reports  , DOI: ( /j.celrep )
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