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Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome.

Mukherjee K, Siddiqi SA, Hashim S, Raje M, Basu SK, Mukhopadhyay A - J. Cell Biol. (2000)

Bottom Line: LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7.Binding of NSF with LSP required prior presence of rab5 on the phagosome.We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.

ABSTRACT
To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPgammaS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)-depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPgammaS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of alpha-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.

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Binding of NSF and rab5 on different phagosomes. (Top) Phagosomes stripped of endogenous NSF by 0.5 M KCl treatment were incubated in presence of purified NSF in the fusion buffer containing cytosol as described in Materials and Methods. Presence of NSF on the phagosomes was determined by Western blot using specific antibody using ECL. Phagosomes were treated with Rab-GDI to remove endogenous rab5 and NSF and incubated in presence of purified NSF, rab5, or rab7 in the fusion buffer containing cytosol as described in Materials and Methods. Presence of NSF (second row), rab5 (third row), and rab7 (bottom) on respective phagosomes was determined using specific antibodies by Western blot using ECL. LSP, live Salmonella-containing phagosome; CF-LSP, LSP treated with ciprofloxacin; DSP, dead Salmonella-containing phagosome.
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Figure 7: Binding of NSF and rab5 on different phagosomes. (Top) Phagosomes stripped of endogenous NSF by 0.5 M KCl treatment were incubated in presence of purified NSF in the fusion buffer containing cytosol as described in Materials and Methods. Presence of NSF on the phagosomes was determined by Western blot using specific antibody using ECL. Phagosomes were treated with Rab-GDI to remove endogenous rab5 and NSF and incubated in presence of purified NSF, rab5, or rab7 in the fusion buffer containing cytosol as described in Materials and Methods. Presence of NSF (second row), rab5 (third row), and rab7 (bottom) on respective phagosomes was determined using specific antibodies by Western blot using ECL. LSP, live Salmonella-containing phagosome; CF-LSP, LSP treated with ciprofloxacin; DSP, dead Salmonella-containing phagosome.

Mentions: To determine the binding of NSF to the phagosomes, KCl-treated LSP, CF-LSP, and DSP were incubated in the presence of purified NSF in fusion buffer containing cytosol for 10 min at 37°C. Treatment of the phagosomes by 0.5 M KCl selectively stripped the endogenous NSF leaving the rab5 on the phagosomal membrane as revealed by Western blot analysis (data not shown). The result presented in the Fig. 7 (upper panel) shows efficient binding of NSF by the LSP. No detectable binding of NSF was observed with CF-LSP or with DSP.


Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome.

Mukherjee K, Siddiqi SA, Hashim S, Raje M, Basu SK, Mukhopadhyay A - J. Cell Biol. (2000)

Binding of NSF and rab5 on different phagosomes. (Top) Phagosomes stripped of endogenous NSF by 0.5 M KCl treatment were incubated in presence of purified NSF in the fusion buffer containing cytosol as described in Materials and Methods. Presence of NSF on the phagosomes was determined by Western blot using specific antibody using ECL. Phagosomes were treated with Rab-GDI to remove endogenous rab5 and NSF and incubated in presence of purified NSF, rab5, or rab7 in the fusion buffer containing cytosol as described in Materials and Methods. Presence of NSF (second row), rab5 (third row), and rab7 (bottom) on respective phagosomes was determined using specific antibodies by Western blot using ECL. LSP, live Salmonella-containing phagosome; CF-LSP, LSP treated with ciprofloxacin; DSP, dead Salmonella-containing phagosome.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169378&req=5

Figure 7: Binding of NSF and rab5 on different phagosomes. (Top) Phagosomes stripped of endogenous NSF by 0.5 M KCl treatment were incubated in presence of purified NSF in the fusion buffer containing cytosol as described in Materials and Methods. Presence of NSF on the phagosomes was determined by Western blot using specific antibody using ECL. Phagosomes were treated with Rab-GDI to remove endogenous rab5 and NSF and incubated in presence of purified NSF, rab5, or rab7 in the fusion buffer containing cytosol as described in Materials and Methods. Presence of NSF (second row), rab5 (third row), and rab7 (bottom) on respective phagosomes was determined using specific antibodies by Western blot using ECL. LSP, live Salmonella-containing phagosome; CF-LSP, LSP treated with ciprofloxacin; DSP, dead Salmonella-containing phagosome.
Mentions: To determine the binding of NSF to the phagosomes, KCl-treated LSP, CF-LSP, and DSP were incubated in the presence of purified NSF in fusion buffer containing cytosol for 10 min at 37°C. Treatment of the phagosomes by 0.5 M KCl selectively stripped the endogenous NSF leaving the rab5 on the phagosomal membrane as revealed by Western blot analysis (data not shown). The result presented in the Fig. 7 (upper panel) shows efficient binding of NSF by the LSP. No detectable binding of NSF was observed with CF-LSP or with DSP.

Bottom Line: LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7.Binding of NSF with LSP required prior presence of rab5 on the phagosome.We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.

ABSTRACT
To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPgammaS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)-depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPgammaS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of alpha-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.

Show MeSH