Journal: PLoS ONE
Article Title: Unconventional Secretion of Tissue Transglutaminase Involves Phospholipid-Dependent Delivery into Recycling Endosomes
doi: 10.1371/journal.pone.0019414
Figure Lengend Snippet: (A) Binding of tTG to recycling endosomes. The amounts of purified 125 I-tTG bound at 4°C to recycling endosomes immunoisolated from NIH3T3 cells lacking tTG were determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (B) The role of phosphoinositides in the tTG interaction with recycling endosomes. Binding of 50 nM purified tTG to recycling endosomes was tested in the presence of 100 µM free phosphoinositides. Bound tTG and endosomal Rab11A/Rab11B and transferrin receptor (TfR) were detected by immuno-blotting. The relative amounts of vesicle-bound tTG were quantified and compared to that in the sample without phosphoinositides in four independent experiments. Bars depict means ± SEM, *p<0.05. (C) The putative phospholipid-binding sequence and site within the fourth domain of tTG. (D) Comparative analysis of tTG binding to phosphoinositides. 125 I-tTG binding to synthetic liposomes (PolyPIPosomes™) containing indicated phiosphoinositides was determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (E) Association of tTG with phosphoinositides in cells. tTG was immunoprecipitated from extracts of WI-38 fibroblasts and the immune complexes were probed by immunoblotting for PI(3)P or PI(4)P. (F) The phospholipid-binding site in tTG is required for membrane targeting and externalization of the protein. K598A,K600A,R601A,K602A mutations (m-plbs) were generated within the phospholipid-binding site of tTG. NIH3T3 fibroblasts transfected with wild type tTG (wt) or its mutant deficient in phosphoinositide binding (m-plbs) were left untreated or treated with 10 mM neomycin for 24 h before induction of these proteins for 4 h. The contents of wt and m-plbs tTG in the recycling endosomes and cytosol were defined by immunoblotting ( left panels ). Cell surface and total levels of wt and m-plbs tTG were defined by surface biotinylation, isolation of surface protein fractions, and immunoblotting ( right panels ). The relative contents of wt and m-plbs tTG in the recycling endosomes and on the cell surface were quantified and compared to those for untreated cells expressing wt protein. Shown are representative of three independent experiments. Bars depict means ± SEM, **p<0.005. See also . (G) A proposed mechanism of unconventional tTG secretion. The de novo synthesized intracellular (cytoplasmic) tTG ( hexagons ) does not follow the classical, ER/Golgi-dependent secretion pathway, but is exported via a four-step unconventional secretion process mediated by recycling endosomes ( dotted arrows ). Solid arrows mark the major intracellular recycling route through the PNRC utilized by β1 integrins. See further comments in the text.
Article Snippet: Membrane lipid strips (#P6002); PIP strips (#P6001), PolyPIPosomes (#YP000; #YP003; #YP004; #YP005; #YP034; #YP035; #YP045; #YP039), and purified synthetic phosphoinositides were from Echelon Biosciences.
Techniques: Binding Assay, Purification, Sequencing, Immunoprecipitation, Western Blot, Generated, Transfection, Mutagenesis, Isolation, Expressing, Synthesized
Echelon Biosciences is a verified supplier 
Echelon Biosciences manufactures this product 
