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Vesicular transport of a ribonucleoprotein to mitochondria.

Mukherjee J, Mahato B, Adhya S - Biol Open (2014)

Bottom Line: In vitro, RNP was directly transferred from the Type 2 vesicles to mitochondria.Live-cell imaging captured spherical Cav1(-) RNP vesicles emerging from the fission of large Cav(+) particles.Thus, RNP appears to traffic by a different route than the classical Rab5-dependent pathway of viral transport.

View Article: PubMed Central - PubMed

Affiliation: Genetic Engineering Laboratory, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India Present address: Penn Institute for Regenerative Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA.

No MeSH data available.


Related in: MedlinePlus

Transfer of RNP from cytosolic vesicles to mitochondria in vitro.(A) Transfer of pcRNA1 from post-caveosomal vesicles to mitochondria in vitro. The RNP-loaded cytosolic fraction (Cyto (R), 10 µl) from 2 h-pcRNA1-transfected FLP32.39 cells was incubated with mitochondria from untreated cells in the presence of ATP or GTP as indicated; Cav− (R), Cyto (R) fraction immunodepleted of Cav1+ vesicles. After incubation, the reactions were centrifuged to separate the supernatant (C) and mitochondrial (M) fractions which were analyzed by Northern blot using anti-COII probe. In lanes 10–12, the Cyto (R) fraction was pre-incubated with the indicated antibody (1:200) before incubation with mitochondria. In lane 13, Cyto(R) was replaced by Cav−(R). Alternatively, mitochondria were pre-incubated with CCCP (50 µM, 10 min, 4°C) before incubation with Cyto(R) (lanes 14, 15). Re-isolated CCCP-treated mitochondria were subjected to freeze thaw lysis followed by centrifugal separation of the soluble matrix (Mx, lane 14) and insoluble membrane (Mb, lane 15) fractions. (B) The 2 h- cytosolic fraction from HepG2 cells transfected with RIC was incubated with mitochondria in the absence (lanes 1–3) or presence (lanes 3–6) of ATP, the mitochondria were re-isolated, solubilised with Triton X-100, and subjected to IP with the indicated antibodies. Immunoprecipitates were probed with a mixture of anti-RIC1, anti-RIC4A and anti-RIC8B. (C) The 2-h cytosolic fraction from normal (WT) or EEA1-KD cells transfected with GFP RNA was incubated with mitochondria under transfer conditions and the soluble (C2) and mitochondrial (M) compartments reisolated for Northern Blot.
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f06: Transfer of RNP from cytosolic vesicles to mitochondria in vitro.(A) Transfer of pcRNA1 from post-caveosomal vesicles to mitochondria in vitro. The RNP-loaded cytosolic fraction (Cyto (R), 10 µl) from 2 h-pcRNA1-transfected FLP32.39 cells was incubated with mitochondria from untreated cells in the presence of ATP or GTP as indicated; Cav− (R), Cyto (R) fraction immunodepleted of Cav1+ vesicles. After incubation, the reactions were centrifuged to separate the supernatant (C) and mitochondrial (M) fractions which were analyzed by Northern blot using anti-COII probe. In lanes 10–12, the Cyto (R) fraction was pre-incubated with the indicated antibody (1:200) before incubation with mitochondria. In lane 13, Cyto(R) was replaced by Cav−(R). Alternatively, mitochondria were pre-incubated with CCCP (50 µM, 10 min, 4°C) before incubation with Cyto(R) (lanes 14, 15). Re-isolated CCCP-treated mitochondria were subjected to freeze thaw lysis followed by centrifugal separation of the soluble matrix (Mx, lane 14) and insoluble membrane (Mb, lane 15) fractions. (B) The 2 h- cytosolic fraction from HepG2 cells transfected with RIC was incubated with mitochondria in the absence (lanes 1–3) or presence (lanes 3–6) of ATP, the mitochondria were re-isolated, solubilised with Triton X-100, and subjected to IP with the indicated antibodies. Immunoprecipitates were probed with a mixture of anti-RIC1, anti-RIC4A and anti-RIC8B. (C) The 2-h cytosolic fraction from normal (WT) or EEA1-KD cells transfected with GFP RNA was incubated with mitochondria under transfer conditions and the soluble (C2) and mitochondrial (M) compartments reisolated for Northern Blot.

Mentions: While it is evident that Type 1 and Type 2 vesicles are present prior to the entry of RNP into mitochondria, it remained to be determined if such vesicles are true intermediates in the transport pathway. Therefore, we isolated the cytosolic fraction from HepG2 cells treated with pcRNA1-RIC complex for 2 h, and incubated it in presence of nucleotide co-factors with mitochondria from untreated FLP32.39 cells. The mitochondria were re-isolated by centrifugation and analyzed for the presence of RNA. Under these conditions the RNA was transferred from the cytosolic fraction to the mitochondria; this was evident from the appearance of COII mRNA by processing of pcRNA1 (Fig. 6A, lane 2). In control reactions lacking mitochondria, the RNA remained in the supernatant after centrifugation (Fig. 6A, lane 3), ruling out non-specific aggregation of the RNA-loaded vesicles during incubation. Transfer required ATP but not GTP (Fig. 6A, lanes 6, 8). Transfer was blocked by antibody against the subunits RIC4A or RIC8B, but not by anti Cav1 antibody (Fig. 6A, lanes 10–12). Since the 2 h post-mitochondrial fraction contains Type 1 as well as Type 2 vesicles (Fig. 4), we examined which of these was involved in the targeting process. Type 2 vesicles, isolated by immunodepletion of the Cav+ Type 1 vesicles, was as effective as the cytosolic fraction in the targeting assay, indicating these to be the true targeting vesicles (Fig. 6A, lane 13). In presence of the mitochondrial uncoupler CCCP, transfer to the mitochondrial membrane was not affected, but the pcRNA was bound to the mitochondrial membrane and not imported into the matrix, and hence not processed (Fig. 6A, lanes 14, 15). This is in keeping with the well known requirement of RIC-catalyzed RNA import for a transmembrane proton gradient (Bhattacharyya and Adhya, 2004).


Vesicular transport of a ribonucleoprotein to mitochondria.

Mukherjee J, Mahato B, Adhya S - Biol Open (2014)

Transfer of RNP from cytosolic vesicles to mitochondria in vitro.(A) Transfer of pcRNA1 from post-caveosomal vesicles to mitochondria in vitro. The RNP-loaded cytosolic fraction (Cyto (R), 10 µl) from 2 h-pcRNA1-transfected FLP32.39 cells was incubated with mitochondria from untreated cells in the presence of ATP or GTP as indicated; Cav− (R), Cyto (R) fraction immunodepleted of Cav1+ vesicles. After incubation, the reactions were centrifuged to separate the supernatant (C) and mitochondrial (M) fractions which were analyzed by Northern blot using anti-COII probe. In lanes 10–12, the Cyto (R) fraction was pre-incubated with the indicated antibody (1:200) before incubation with mitochondria. In lane 13, Cyto(R) was replaced by Cav−(R). Alternatively, mitochondria were pre-incubated with CCCP (50 µM, 10 min, 4°C) before incubation with Cyto(R) (lanes 14, 15). Re-isolated CCCP-treated mitochondria were subjected to freeze thaw lysis followed by centrifugal separation of the soluble matrix (Mx, lane 14) and insoluble membrane (Mb, lane 15) fractions. (B) The 2 h- cytosolic fraction from HepG2 cells transfected with RIC was incubated with mitochondria in the absence (lanes 1–3) or presence (lanes 3–6) of ATP, the mitochondria were re-isolated, solubilised with Triton X-100, and subjected to IP with the indicated antibodies. Immunoprecipitates were probed with a mixture of anti-RIC1, anti-RIC4A and anti-RIC8B. (C) The 2-h cytosolic fraction from normal (WT) or EEA1-KD cells transfected with GFP RNA was incubated with mitochondria under transfer conditions and the soluble (C2) and mitochondrial (M) compartments reisolated for Northern Blot.
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Related In: Results  -  Collection

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f06: Transfer of RNP from cytosolic vesicles to mitochondria in vitro.(A) Transfer of pcRNA1 from post-caveosomal vesicles to mitochondria in vitro. The RNP-loaded cytosolic fraction (Cyto (R), 10 µl) from 2 h-pcRNA1-transfected FLP32.39 cells was incubated with mitochondria from untreated cells in the presence of ATP or GTP as indicated; Cav− (R), Cyto (R) fraction immunodepleted of Cav1+ vesicles. After incubation, the reactions were centrifuged to separate the supernatant (C) and mitochondrial (M) fractions which were analyzed by Northern blot using anti-COII probe. In lanes 10–12, the Cyto (R) fraction was pre-incubated with the indicated antibody (1:200) before incubation with mitochondria. In lane 13, Cyto(R) was replaced by Cav−(R). Alternatively, mitochondria were pre-incubated with CCCP (50 µM, 10 min, 4°C) before incubation with Cyto(R) (lanes 14, 15). Re-isolated CCCP-treated mitochondria were subjected to freeze thaw lysis followed by centrifugal separation of the soluble matrix (Mx, lane 14) and insoluble membrane (Mb, lane 15) fractions. (B) The 2 h- cytosolic fraction from HepG2 cells transfected with RIC was incubated with mitochondria in the absence (lanes 1–3) or presence (lanes 3–6) of ATP, the mitochondria were re-isolated, solubilised with Triton X-100, and subjected to IP with the indicated antibodies. Immunoprecipitates were probed with a mixture of anti-RIC1, anti-RIC4A and anti-RIC8B. (C) The 2-h cytosolic fraction from normal (WT) or EEA1-KD cells transfected with GFP RNA was incubated with mitochondria under transfer conditions and the soluble (C2) and mitochondrial (M) compartments reisolated for Northern Blot.
Mentions: While it is evident that Type 1 and Type 2 vesicles are present prior to the entry of RNP into mitochondria, it remained to be determined if such vesicles are true intermediates in the transport pathway. Therefore, we isolated the cytosolic fraction from HepG2 cells treated with pcRNA1-RIC complex for 2 h, and incubated it in presence of nucleotide co-factors with mitochondria from untreated FLP32.39 cells. The mitochondria were re-isolated by centrifugation and analyzed for the presence of RNA. Under these conditions the RNA was transferred from the cytosolic fraction to the mitochondria; this was evident from the appearance of COII mRNA by processing of pcRNA1 (Fig. 6A, lane 2). In control reactions lacking mitochondria, the RNA remained in the supernatant after centrifugation (Fig. 6A, lane 3), ruling out non-specific aggregation of the RNA-loaded vesicles during incubation. Transfer required ATP but not GTP (Fig. 6A, lanes 6, 8). Transfer was blocked by antibody against the subunits RIC4A or RIC8B, but not by anti Cav1 antibody (Fig. 6A, lanes 10–12). Since the 2 h post-mitochondrial fraction contains Type 1 as well as Type 2 vesicles (Fig. 4), we examined which of these was involved in the targeting process. Type 2 vesicles, isolated by immunodepletion of the Cav+ Type 1 vesicles, was as effective as the cytosolic fraction in the targeting assay, indicating these to be the true targeting vesicles (Fig. 6A, lane 13). In presence of the mitochondrial uncoupler CCCP, transfer to the mitochondrial membrane was not affected, but the pcRNA was bound to the mitochondrial membrane and not imported into the matrix, and hence not processed (Fig. 6A, lanes 14, 15). This is in keeping with the well known requirement of RIC-catalyzed RNA import for a transmembrane proton gradient (Bhattacharyya and Adhya, 2004).

Bottom Line: In vitro, RNP was directly transferred from the Type 2 vesicles to mitochondria.Live-cell imaging captured spherical Cav1(-) RNP vesicles emerging from the fission of large Cav(+) particles.Thus, RNP appears to traffic by a different route than the classical Rab5-dependent pathway of viral transport.

View Article: PubMed Central - PubMed

Affiliation: Genetic Engineering Laboratory, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India Present address: Penn Institute for Regenerative Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA.

No MeSH data available.


Related in: MedlinePlus