Limits...
The polybasic region of Rac1 modulates bacterial uptake independently of self-association and membrane targeting.

Wong KW, Mohammadi S, Isberg RR - J. Biol. Chem. (2008)

Bottom Line: To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail.The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association.This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase member, is required for Rac1 self-association, membrane localization, nuclear translocation, and interaction with downstream effectors. We previously demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the effectors that requires the polybasic region for interaction, is necessary for efficient invasin-promoted uptake of Yersinia pseudotuberculosis by nonphagocytic cells. Here we further examined the role of this region in invasin-promoted uptake. Using fluorescence resonance energy transfer experiments (FRET), we determined that engagement of integrin receptors by invasin caused elevated levels of Rac1 self-association at the site of bacterial adhesion in a PBR-dependent fashion. Self-association could be disrupted using several strategies: translocation of the Yersinia YopT prenylcysteine protease into host cells, inactivation of the Rac1 isoprenylation signal that is required for membrane localization, and elimination of the PBR. Disruption in each case impaired invasin-promoted uptake. To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail. The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association. This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.

Show MeSH

Related in: MedlinePlus

Rac1 self-association is stimulated by Yersinia infection. COS1 cells were transfected with mCFP-Rac1 and mYFP-Rac1 constructs (A and D), mCFP-GerGer and mYFP-GerGer (B), or Lyn-mCFP-Rac1(C189S) and Lyn-mYFP-Rac1(C189S) (C). Transfected cells were challenged the next day with a 30-min incubation of YPIII(p-) (A-C) or YP17/pYopT (D), followed by immunostaining of extracellular bacteria (Ext. bacteria) bound onto host cells. Sensitized and normalized FRET readings (sens. FRET and norm. FRET, respectively) were determined as described (see “Materials and Methods”). The arrows denote nascent phagosomes. E, mean normalized FRET levels at nascent phagosomes were plotted comparing mCFP-Rac1/mYFP-Rac1 and mCFP-GerGer/mYFP-GerGer as described in the legend to Fig. 3I. White scale bar in D, 5 μm (applies to all panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2602895&req=5

fig7: Rac1 self-association is stimulated by Yersinia infection. COS1 cells were transfected with mCFP-Rac1 and mYFP-Rac1 constructs (A and D), mCFP-GerGer and mYFP-GerGer (B), or Lyn-mCFP-Rac1(C189S) and Lyn-mYFP-Rac1(C189S) (C). Transfected cells were challenged the next day with a 30-min incubation of YPIII(p-) (A-C) or YP17/pYopT (D), followed by immunostaining of extracellular bacteria (Ext. bacteria) bound onto host cells. Sensitized and normalized FRET readings (sens. FRET and norm. FRET, respectively) were determined as described (see “Materials and Methods”). The arrows denote nascent phagosomes. E, mean normalized FRET levels at nascent phagosomes were plotted comparing mCFP-Rac1/mYFP-Rac1 and mCFP-GerGer/mYFP-GerGer as described in the legend to Fig. 3I. White scale bar in D, 5 μm (applies to all panels).

Mentions: To determine whether plasma membrane localization was sufficient to cause intermolecular FRET in this particular assay, we targeted mCFP/mYFP onto the plasma membrane by either an NH2-terminal myristoylation signal from the Lyn protein (24) or the COOH-terminal geranylgeranylation site from Rac1 (see Fig. 3H for Lyn-mYFP localization). Neither of the two tags could enable the CFP/YFP pair to generate FRET (Fig. 4, compare A with B and C; see Fig. 5, A-C and I) even at high concentrations (Fig. 5, B and C). This lowered self-association was observed, in addition, with cells that were challenged with Y. pseudotuberculosis, since no FRET signal could be detected at regions of membrane aggregation resulting from invasin binding or at regions of membrane ruffling (see below; Fig. 7, B and C). We conclude that plasma membrane localization was not sufficient to cause protein self-association.


The polybasic region of Rac1 modulates bacterial uptake independently of self-association and membrane targeting.

Wong KW, Mohammadi S, Isberg RR - J. Biol. Chem. (2008)

Rac1 self-association is stimulated by Yersinia infection. COS1 cells were transfected with mCFP-Rac1 and mYFP-Rac1 constructs (A and D), mCFP-GerGer and mYFP-GerGer (B), or Lyn-mCFP-Rac1(C189S) and Lyn-mYFP-Rac1(C189S) (C). Transfected cells were challenged the next day with a 30-min incubation of YPIII(p-) (A-C) or YP17/pYopT (D), followed by immunostaining of extracellular bacteria (Ext. bacteria) bound onto host cells. Sensitized and normalized FRET readings (sens. FRET and norm. FRET, respectively) were determined as described (see “Materials and Methods”). The arrows denote nascent phagosomes. E, mean normalized FRET levels at nascent phagosomes were plotted comparing mCFP-Rac1/mYFP-Rac1 and mCFP-GerGer/mYFP-GerGer as described in the legend to Fig. 3I. White scale bar in D, 5 μm (applies to all panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602895&req=5

fig7: Rac1 self-association is stimulated by Yersinia infection. COS1 cells were transfected with mCFP-Rac1 and mYFP-Rac1 constructs (A and D), mCFP-GerGer and mYFP-GerGer (B), or Lyn-mCFP-Rac1(C189S) and Lyn-mYFP-Rac1(C189S) (C). Transfected cells were challenged the next day with a 30-min incubation of YPIII(p-) (A-C) or YP17/pYopT (D), followed by immunostaining of extracellular bacteria (Ext. bacteria) bound onto host cells. Sensitized and normalized FRET readings (sens. FRET and norm. FRET, respectively) were determined as described (see “Materials and Methods”). The arrows denote nascent phagosomes. E, mean normalized FRET levels at nascent phagosomes were plotted comparing mCFP-Rac1/mYFP-Rac1 and mCFP-GerGer/mYFP-GerGer as described in the legend to Fig. 3I. White scale bar in D, 5 μm (applies to all panels).
Mentions: To determine whether plasma membrane localization was sufficient to cause intermolecular FRET in this particular assay, we targeted mCFP/mYFP onto the plasma membrane by either an NH2-terminal myristoylation signal from the Lyn protein (24) or the COOH-terminal geranylgeranylation site from Rac1 (see Fig. 3H for Lyn-mYFP localization). Neither of the two tags could enable the CFP/YFP pair to generate FRET (Fig. 4, compare A with B and C; see Fig. 5, A-C and I) even at high concentrations (Fig. 5, B and C). This lowered self-association was observed, in addition, with cells that were challenged with Y. pseudotuberculosis, since no FRET signal could be detected at regions of membrane aggregation resulting from invasin binding or at regions of membrane ruffling (see below; Fig. 7, B and C). We conclude that plasma membrane localization was not sufficient to cause protein self-association.

Bottom Line: To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail.The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association.This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.

ABSTRACT
The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase member, is required for Rac1 self-association, membrane localization, nuclear translocation, and interaction with downstream effectors. We previously demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the effectors that requires the polybasic region for interaction, is necessary for efficient invasin-promoted uptake of Yersinia pseudotuberculosis by nonphagocytic cells. Here we further examined the role of this region in invasin-promoted uptake. Using fluorescence resonance energy transfer experiments (FRET), we determined that engagement of integrin receptors by invasin caused elevated levels of Rac1 self-association at the site of bacterial adhesion in a PBR-dependent fashion. Self-association could be disrupted using several strategies: translocation of the Yersinia YopT prenylcysteine protease into host cells, inactivation of the Rac1 isoprenylation signal that is required for membrane localization, and elimination of the PBR. Disruption in each case impaired invasin-promoted uptake. To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail. The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association. This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors.

Show MeSH
Related in: MedlinePlus