Journal: Journal of Cell Science

Article Title: Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells

doi: 10.1242/jcs.200204

Figure Lengend Snippet: Targeting survey for TA proteins in mammalian cells. (A) Subcellular localization patterns for selected TA proteins. COS-7 cells transfected with Myc–ACBD5, Myc–FALDH-PO, Myc–FALDH-ER, GFP–BCL-XL or GFP–BAK were immunolabeled using anti-PEX14 (PO) and anti-Myc antibodies. Arrows highlight regions of colocalization (BCL-XL) or lack of colocalization (BAK, FALDH-ER) with peroxisomes. Higher magnifications of boxed regions are shown. Scale bars: 10 µm (overview), 5 µm (overlay). (B) Table summarizing the TA proteins analyzed. MITO, mitochondria; PO, peroxisomes.

Article Snippet: Fluorescently labeled peptides ACBD5 WT (FITC–SPGVLTFAIIWPFIAQWLVYLYYQRRRRKL), MUT1 (FITC–SPGVLTFAIIWPFIAQWLVYLYYQRARAKL) and MUT2 (FITC–SPGVLTFAIIWPFIAQWLVYLYYQAAAAKL) (Genscript) were used in the assay at a final concentration of 6.7 nM.

Techniques: Transfection, Immunolabeling

Journal: Journal of Cell Science

Article Title: Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells

doi: 10.1242/jcs.200204

Figure Lengend Snippet: Alterations in tail charge and TMD GRAVY redistribute TA proteins to other organelles. (A) Domain structure of ACBD5, and the GFP–ACBD5 TMD-T WT and mutant (MUT1–MUT3) constructs used. ACB, acyl CoA-binding domain. (B) COS-7 cells transfected with GFP–ACBD5 TMD-T WT or MUT1–MUT3 and, where indicated, Myc–VAPB (ER), were labeled with anti-PEX14 (for peroxisomes, PO), anti-TOM20 (for mitochondria, MITO) and anti-Myc antibodies. (C) Domain structure of FIS1 WT and SR. TPR, tetratricopeptide repeat domain. (D) COS-7 cells transfected with FLAG fusions of FIS1-WT or FIS1-SR were labeled with anti-TOM20 and anti-FLAG antibodies. (E) Qualitative analysis of GFP–ACBD5 TMD-T (A,B) and FLAG–FIS1 (C,D) localization. A minimum of 300 cells were examined per condition, and organelle localization was microscopically assessed. The percentage of cells with PO, MITO, ER or shared localization is shown (for ACBD5 WT or MUT1 and FIS1-WT, shared is the percentage of cells with both PO and MITO staining; for ACBD5 MUT2, shared is the percentage of cells with MITO and ER staining; for ACBD5 MUT3, shared is the percentage of cells with ER and PO staining). Values represent mean±s.e.m. of three independent experiments. Higher magnification view of boxed regions in B and D is shown. Scale bars: 20 µm (overview), 10 µm (overlay).

Article Snippet: Fluorescently labeled peptides ACBD5 WT (FITC–SPGVLTFAIIWPFIAQWLVYLYYQRRRRKL), MUT1 (FITC–SPGVLTFAIIWPFIAQWLVYLYYQRARAKL) and MUT2 (FITC–SPGVLTFAIIWPFIAQWLVYLYYQAAAAKL) (Genscript) were used in the assay at a final concentration of 6.7 nM.

Techniques: Mutagenesis, Construct, Binding Assay, Transfection, Labeling, Staining

Journal: Journal of Cell Science

Article Title: Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells

doi: 10.1242/jcs.200204

Figure Lengend Snippet: PEX19 affinity is a key determinant in targeting the peroxisomal membrane. (A,B) Immunoblots of co-immunoprecipitations from COS-7 cell lysates from cells expressing HA–PEX19 and GFP fusions as indicated, using GFP-Trap. Cytosolic GFP was used as a control. Input (1% of total), total cell lysates; IP, immunoprecipitation. A dotted line in A indicates where a region of gel is not shown for better visualization. (C) Normalized representative curves of fluorescence anisotropy measurements, using recombinant PEX19 and fluorescently labeled peptides (ACBD5-TMD-T) (see A). Average K d (µM) values were WT, 0.9±0.5; MUT1, 1.9±0.5; MUT2, 7.7±0.2. Values represent mean±s.d. of three independent measurements. (D) Control and PEX19-deficient fibroblasts transfected with GFP and FLAG fusions as indicated were labeled with anti-PEX14, anti-TOM20 and anti-FLAG antibodies. Higher magnification view of boxed regions is shown. Scale bars: 20 µm (top panel), 5 µm (lower panels). (E) mRNAs for ACBD5 TMD-T constructs, PEX26 TMD-T and SEC61β TMD-T were in vitro translated in the presence of recombinant Nc PEX19 or GET3 (5 µM) and aggregation was monitored by using fluorescence microscopy. Scale bar: 2 µm. (F) Immunoblots showing levels of in vitro -translated proteins. Equal amounts of a representative translation reaction were loaded and the blot probed with an anti-HA antibody; a band from a Coomassie-stained gel run in parallel serves as a loading control. (G) Solubilizing activity as determined by quantification of aggregate number with data from 10 individual fields of view. Values represent mean±s.e.m. of three independent experiments. *** P <0.001; ns, not significant compared to the indicated group (unpaired t -test).

Article Snippet: Fluorescently labeled peptides ACBD5 WT (FITC–SPGVLTFAIIWPFIAQWLVYLYYQRRRRKL), MUT1 (FITC–SPGVLTFAIIWPFIAQWLVYLYYQRARAKL) and MUT2 (FITC–SPGVLTFAIIWPFIAQWLVYLYYQAAAAKL) (Genscript) were used in the assay at a final concentration of 6.7 nM.

Techniques: Membrane, Western Blot, Expressing, Control, Immunoprecipitation, Fluorescence, Recombinant, Labeling, Transfection, Construct, In Vitro, Microscopy, Staining, Activity Assay