Journal: PLOS Biology

Article Title: SLMO transfers phosphatidylserine between the outer and inner mitochondrial membrane in Drosophila

doi: 10.1371/journal.pbio.3002941

Figure Lengend Snippet: (A) SLMO transfer PS between liposomes. A schematic diagram of the lipid transfer assay is shown on the left, in which donor liposomes with NBD-PS or NBD-PE, and acceptor liposomes were incubated with MBP-SLMO. Time courses of normalized fluorescence signals from liposome mixtures containing NBD-PS or NBD-PE in the donor liposomes with SLMO or BSA are plotted. (B) Design of mutant SLMO in PS harboring. From MD calculation, Thr93 and Asn150 interact with PS, thus mutating both Thr93 and Asn150 to Ala may prevent PS binding in the pocket. (C) SLMO T93A and SLMO N150A abolish the PS transporting ability. Time courses of normalized fluorescence signals from liposome mixtures containing NBD-PS in the donor liposomes with SLMO, SLMO T93A , or SLMO N150A are shown. (D, E) SLMO T93A and SLMO N150A are not able to replace SLMO in promoting mitochondrial organization and cellular function. (D) Muscles sections of control ( MHC-gal4/UAS-GFP RNAi ), slmo RNAi1 ( MHC-gal4/UAS-slmo RNAi1 ), slmo RNAi1 +EGFP ( MHC-gal4/+;UAS-slmo RNAi1 /UAS-GFP ), slmo RNAi1 +slmo R1 ( MHC-gal4/+;UAS-slmo RNAi1 /DA-slmo R1 ), slmo RNAi1 +slmo T93A ( MHC-gal4/+;UAS-slmo RNAi1 /DA-slmo T93A ), and slmo RNAi1 +slmo N150A ( MHC-gal4/+;UAS-slmo RNAi1 /DA-slmo N150A ) flies. M, Mitochondria; F, myofibrils. Scale bar, 1 μm. (E) Quantification of ERG amplitudes and off-transients of control ( GMR-gal4/UAS-GFP RNAi ), slmo RNAi1 ( GMR-gal4/UAS-slmo RNAi1 ), slmo RNAi1 +EGFP ( GMR-gal4/+;UAS-slmo RNAi1 /UAS-GFP ), slmo RNAi1 +slmo T93A ( GMR-gal4/+;UAS-slmo RNAi1 /UAS-slmo T93A ), slmo RNAi1 +slmo N150A ( GMR-gal4/+;UAS-slmo RNAi1 /DA-slmo N150A ), and slmo RNAi1 +slmo R1 ( GMR-gal4/+;UAS-slmo RNAi1 /DA-slmo R1 ) flies. ERG from at least 10 flies was used, and significant differences were determined using the unpaired t test. DA-slmo R1 , DA-slmo T93A , and DA-slmo N150A were designed as slmo RNAi1 resistant. (F) Lipidomic analysis of mitochondrial PE and PS levels of control ( MHC-gal4/GFP RNAi ), pisd RNAi ( MHC-gal4/UAS-pisd RNAi ), slmo RNAi1 ( MHC-gal4/UAS-slmo RNAi1 ), and slmo RNAi + slmo R1 ( MHC-gal4/+;UAS-slmo RNAi /UAS-slmo R1 ) muscles. Mitochondria were isolated from 10 dissected thoraxes per assay, and 4 replicates were quantified. (G) Knocking down slmo in S2 cells increased PS levels in the OMM. Live images of cells transfected with Tom20-PSS (red), GFP-LactC1C2 (green), and dsRNA of BFP (control) or slmo ( slmo RNAi ). Scale bar, 10 μm. Manders’ Colocalization Coefficients (MCC) of GFP-LactC1C2 and Tom20-RFP of S2 cells transfected with BFP RNAi , TOM20-PSS, slmo RNAi pss RNAi , or pisd RNAi were quantified by ImageJ. The data underlying the graphs shown in the figure can be found in . ERG, electroretinogram; MD, molecular dynamics; OMM, outer mitochondrial membrane; PE, phosphatidylethanolamine; PS, phosphatidylserine.

Article Snippet: The slmo , slmo T93A , and slmo N150A cDNA were subcloned into the pMAL-c6T Vector (New England Biolabs, E8201S) between the BamHI and EcoRI sites, and the plasmid was transformed into BL21 (DE3) competent cells (TransGen Biotech, CD601-02).

Techniques: Liposomes, Incubation, Fluorescence, Mutagenesis, Binding Assay, Cell Function Assay, Muscles, Control, Isolation, Transfection, Membrane