Figure 1. KRS secretion via exosomes.... | Rockefeller University Press

Figure 1.

$Figure 1. KRS secretion via exosomes. (A) Human cytosolic KRS (597 aa) consists of anticodon-binding (ABD; 70–214 aa) and catalytic domains (CDs; 222–574 aa; Guo et al., 2008). The caspase-8 cleavage site (VxxD) and PDZ-binding motif (VGTSV) are located in the N- (9–12 aa) and C-terminal (594–597 aa) ends. (B) HCT116 cells were incubated in normal and starvation (Starv.) conditions for 24 h. The culture media were subjected to ultracentrifugation at 100,000 g to obtain the pellet. KRS present in the pellets (containing vesicle fraction) was separated by 8% PAGE and detected by immunoblotting with anti-KRS antibody. Notice a slight difference in the gel mobility of KRS in the medium and cell lysates (see Fig. 3 A for further investigation). (C) Isolated vesicles from B were separated by OptiPrep gradient centrifugation. The nine fractions in the 1.02–1.19 g/ml density range were collected, and the presence of KRS and syntenin (Synt) was analyzed by immunoblotting with their specific antibodies. (D) Dynamic light scattering demonstrated the size distribution of the isolated vesicles (mean diameter, 147.3 nm). (E) Negatively stained field of the KRS-containing vesicles isolated from HCT116 cells as in B. (F and G) To see whether Rab (F) and ESCRT-III (G) proteins are involved in the exosomic secretion of KRS, we transfected HCT116 cells with siRNAs specifically targeting each of the indicated Rab and ESCRT-III proteins and examined whether KRS secretion was affected by the suppression of Rab and ESCRT-III proteins. The secretion of KRS was induced by starvation of HCT116 cells for 24 h. After isolation of the secreted exosomes, the amounts of KRS in the obtained exosomes were compared by immunoblotting with anti-KRS antibody. Quantitation of secreted KRS blots was measured by ImageJ. si-con, control siRNA; WCL, whole-cell lysate. (H and I) Localization of KRS (H) and CD63 (I), a known membrane-bound marker for exosomes, was shown by immunogold-staining EM with their respective antibodies. (J) The isolated vesicles were incubated with or without 0.025% trypsin and 0.1% Triton X-100 at 37°C for 30 min, and the presence of KRS, syntenin, and CD63 was analyzed by immunoblotting with their specific antibodies. (K–M) Immunogold-staining EM shows KRS located in the lumen of the exosome near the target macrophage (K), outside of the exosomes near macrophages (L), and within the target macrophage (M).$

KRS secretion via exosomes. (A) Human cytosolic KRS (597 aa) consists of anticodon-binding (ABD; 70–214 aa) and catalytic domains (CDs; 222–574 aa; Guo et al., 2008). The caspase-8 cleavage site (VxxD) and PDZ-binding motif (VGTSV) are located in the N- (9–12 aa) and C-terminal (594–597 aa) ends. (B) HCT116 cells were incubated in normal and starvation (Starv.) conditions for 24 h. The culture media were subjected to ultracentrifugation at 100,000 g to obtain the pellet. KRS present in the pellets (containing vesicle fraction) was separated by 8% PAGE and detected by immunoblotting with anti-KRS antibody. Notice a slight difference in the gel mobility of KRS in the medium and cell lysates (see Fig. 3 A for further investigation). (C) Isolated vesicles from B were separated by OptiPrep gradient centrifugation. The nine fractions in the 1.02–1.19 g/ml density range were collected, and the presence of KRS and syntenin (Synt) was analyzed by immunoblotting with their specific antibodies. (D) Dynamic light scattering demonstrated the size distribution of the isolated vesicles (mean diameter, 147.3 nm). (E) Negatively stained field of the KRS-containing vesicles isolated from HCT116 cells as in B. (F and G) To see whether Rab (F) and ESCRT-III (G) proteins are involved in the exosomic secretion of KRS, we transfected HCT116 cells with siRNAs specifically targeting each of the indicated Rab and ESCRT-III proteins and examined whether KRS secretion was affected by the suppression of Rab and ESCRT-III proteins. The secretion of KRS was induced by starvation of HCT116 cells for 24 h. After isolation of the secreted exosomes, the amounts of KRS in the obtained exosomes were compared by immunoblotting with anti-KRS antibody. Quantitation of secreted KRS blots was measured by ImageJ. si-con, control siRNA; WCL, whole-cell lysate. (H and I) Localization of KRS (H) and CD63 (I), a known membrane-bound marker for exosomes, was shown by immunogold-staining EM with their respective antibodies. (J) The isolated vesicles were incubated with or without 0.025% trypsin and 0.1% Triton X-100 at 37°C for 30 min, and the presence of KRS, syntenin, and CD63 was analyzed by immunoblotting with their specific antibodies. (K–M) Immunogold-staining EM shows KRS located in the lumen of the exosome near the target macrophage (K), outside of the exosomes near macrophages (L), and within the target macrophage (M).