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VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins.

Swarbrick JD, Shaw DJ, Chhabra S, Ghai R, Valkov E, Norwood SJ, Seaman MN, Collins BM - PLoS ONE (2011)

Bottom Line: VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins.There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case.Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo.

View Article: PubMed Central - PubMed

Affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

ABSTRACT
VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+) and Zn(2+) in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.

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VPS29 binds specifically to SNX1 but with low affinity in vitro.(A) Immunoprecipitations from HeLa cells do not detect association of retromer with SNX1 even in the presence of increased levels of VPS29 expression. Cells expressing GFP-VPS29 or GFP-VPS29(L152E) mutant were subjected to immunoprecipitation with either VPS26 or SNX1 antibodies. VPS26 (and thus VPS29-containing retromer) associates readily with the effector complex containing strumpellin [32], confirming that known binding partners can be detected in the immuno-isolates. However, no SNX1 is detected, and furthermore, in reverse experiments SNX1 does not precipitate retromer indicating that their association in vivo is relatively weak or transient. (B) Titration of VPS29 with SNX1 in NMR experiments reveals specific but weak association in vitro. A selected region is shown for the 15N-HSQC spectra of VPS29 in the presence of increasing concentrations of SNX1. (C) Chemical shift perturbations are shown for VPS29 in the presence of SNX1. Inset shows a plot of the chemical shift perturbation for Leu26 NH as a function of SNX1 concentration. (D) SNX1 binds to VPS29 via the conserved hydrophobic surface on the opposite face to the metal-binding pocket and VPS35 binding interface. Residues that show the largest perturbations on SNX1 binding (>2 standard deviations) are mapped on the VPS29 structure in blue. The structure of VPS29 (surface, and green ribbons) is shown in complex with VPS35(476–780) (red ribbons) [24]. The side-chains of the VPS29 hydrophobic surface are indicated. (E) Mutation of the hydrophobic surface of VPS29 (L152E) prevents VPS29-SNX1 association. The [1H,15N]-HSQC spectra for VPS29(L152E) in the absence (black) and presence (red) of SNX1 indicates no significant association is occurring.
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pone-0020420-g008: VPS29 binds specifically to SNX1 but with low affinity in vitro.(A) Immunoprecipitations from HeLa cells do not detect association of retromer with SNX1 even in the presence of increased levels of VPS29 expression. Cells expressing GFP-VPS29 or GFP-VPS29(L152E) mutant were subjected to immunoprecipitation with either VPS26 or SNX1 antibodies. VPS26 (and thus VPS29-containing retromer) associates readily with the effector complex containing strumpellin [32], confirming that known binding partners can be detected in the immuno-isolates. However, no SNX1 is detected, and furthermore, in reverse experiments SNX1 does not precipitate retromer indicating that their association in vivo is relatively weak or transient. (B) Titration of VPS29 with SNX1 in NMR experiments reveals specific but weak association in vitro. A selected region is shown for the 15N-HSQC spectra of VPS29 in the presence of increasing concentrations of SNX1. (C) Chemical shift perturbations are shown for VPS29 in the presence of SNX1. Inset shows a plot of the chemical shift perturbation for Leu26 NH as a function of SNX1 concentration. (D) SNX1 binds to VPS29 via the conserved hydrophobic surface on the opposite face to the metal-binding pocket and VPS35 binding interface. Residues that show the largest perturbations on SNX1 binding (>2 standard deviations) are mapped on the VPS29 structure in blue. The structure of VPS29 (surface, and green ribbons) is shown in complex with VPS35(476–780) (red ribbons) [24]. The side-chains of the VPS29 hydrophobic surface are indicated. (E) Mutation of the hydrophobic surface of VPS29 (L152E) prevents VPS29-SNX1 association. The [1H,15N]-HSQC spectra for VPS29(L152E) in the absence (black) and presence (red) of SNX1 indicates no significant association is occurring.

Mentions: The final question we have addressed using NMR spectroscopy, is the potential interaction of VPS29 with associated regulatory proteins. Apart from VPS35, VPS29 has also been shown to interact with regulatory proteins; the SNX complex Vps5p/Vps17p in yeast [27], and the Rab GAP TBC1D5 in human cells [32]. Contrasting results have been seen for the binding of either TBC1D5 or the SNX complexes in mammalian cells. TBC1D5 interaction is readily detected in immunoprecipitations of GFP-tagged VPS29 and in yeast 2-hybrid assays, along with other effectors such as the WASH1/strumpellin complex [32] (R. Teasdale, personal communication). The interaction of VPS29 with mammalian SNX proteins is more contentious. To date, we have been unable to detect interactions of VPS29 or the retromer complex with mammalian SNX proteins in similar conditions as those used for TBC1D5 [27], [32]. For example, in experiments shown in Fig. 8A, we over-expressed GFP-VPS29 in HeLa cells in order to enhance potential complex formation, and performed immunoprecipitations with either the core retromer subunit VPS26 or the sorting nexin SNX1. As an initial control we first showed that VPS26 (and hence VPS29-containing retromer) readily binds the protein strumpellin as shown previously [32]. However, no SNX1 is detected in these immunoprecipitations, nor is retromer binding when SNX1 is used as bait, indicating a weak or transient interaction in vivo. The lack of binding is not due to competition by the antibody itself as experiments with epitope-tagged proteins have yielded similar negative results [27], [32]. Although other groups have detected interactions of retromer (both VPS35 and VPS29 subunits) with SNX proteins using ultra-sensitive yeast 2-hybrid assays, or over-expression of all retromer and SNX subunits together [20], [43], [44], there have been no reports of binding between endogenous mammalian proteins.


VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins.

Swarbrick JD, Shaw DJ, Chhabra S, Ghai R, Valkov E, Norwood SJ, Seaman MN, Collins BM - PLoS ONE (2011)

VPS29 binds specifically to SNX1 but with low affinity in vitro.(A) Immunoprecipitations from HeLa cells do not detect association of retromer with SNX1 even in the presence of increased levels of VPS29 expression. Cells expressing GFP-VPS29 or GFP-VPS29(L152E) mutant were subjected to immunoprecipitation with either VPS26 or SNX1 antibodies. VPS26 (and thus VPS29-containing retromer) associates readily with the effector complex containing strumpellin [32], confirming that known binding partners can be detected in the immuno-isolates. However, no SNX1 is detected, and furthermore, in reverse experiments SNX1 does not precipitate retromer indicating that their association in vivo is relatively weak or transient. (B) Titration of VPS29 with SNX1 in NMR experiments reveals specific but weak association in vitro. A selected region is shown for the 15N-HSQC spectra of VPS29 in the presence of increasing concentrations of SNX1. (C) Chemical shift perturbations are shown for VPS29 in the presence of SNX1. Inset shows a plot of the chemical shift perturbation for Leu26 NH as a function of SNX1 concentration. (D) SNX1 binds to VPS29 via the conserved hydrophobic surface on the opposite face to the metal-binding pocket and VPS35 binding interface. Residues that show the largest perturbations on SNX1 binding (>2 standard deviations) are mapped on the VPS29 structure in blue. The structure of VPS29 (surface, and green ribbons) is shown in complex with VPS35(476–780) (red ribbons) [24]. The side-chains of the VPS29 hydrophobic surface are indicated. (E) Mutation of the hydrophobic surface of VPS29 (L152E) prevents VPS29-SNX1 association. The [1H,15N]-HSQC spectra for VPS29(L152E) in the absence (black) and presence (red) of SNX1 indicates no significant association is occurring.
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Related In: Results  -  Collection

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pone-0020420-g008: VPS29 binds specifically to SNX1 but with low affinity in vitro.(A) Immunoprecipitations from HeLa cells do not detect association of retromer with SNX1 even in the presence of increased levels of VPS29 expression. Cells expressing GFP-VPS29 or GFP-VPS29(L152E) mutant were subjected to immunoprecipitation with either VPS26 or SNX1 antibodies. VPS26 (and thus VPS29-containing retromer) associates readily with the effector complex containing strumpellin [32], confirming that known binding partners can be detected in the immuno-isolates. However, no SNX1 is detected, and furthermore, in reverse experiments SNX1 does not precipitate retromer indicating that their association in vivo is relatively weak or transient. (B) Titration of VPS29 with SNX1 in NMR experiments reveals specific but weak association in vitro. A selected region is shown for the 15N-HSQC spectra of VPS29 in the presence of increasing concentrations of SNX1. (C) Chemical shift perturbations are shown for VPS29 in the presence of SNX1. Inset shows a plot of the chemical shift perturbation for Leu26 NH as a function of SNX1 concentration. (D) SNX1 binds to VPS29 via the conserved hydrophobic surface on the opposite face to the metal-binding pocket and VPS35 binding interface. Residues that show the largest perturbations on SNX1 binding (>2 standard deviations) are mapped on the VPS29 structure in blue. The structure of VPS29 (surface, and green ribbons) is shown in complex with VPS35(476–780) (red ribbons) [24]. The side-chains of the VPS29 hydrophobic surface are indicated. (E) Mutation of the hydrophobic surface of VPS29 (L152E) prevents VPS29-SNX1 association. The [1H,15N]-HSQC spectra for VPS29(L152E) in the absence (black) and presence (red) of SNX1 indicates no significant association is occurring.
Mentions: The final question we have addressed using NMR spectroscopy, is the potential interaction of VPS29 with associated regulatory proteins. Apart from VPS35, VPS29 has also been shown to interact with regulatory proteins; the SNX complex Vps5p/Vps17p in yeast [27], and the Rab GAP TBC1D5 in human cells [32]. Contrasting results have been seen for the binding of either TBC1D5 or the SNX complexes in mammalian cells. TBC1D5 interaction is readily detected in immunoprecipitations of GFP-tagged VPS29 and in yeast 2-hybrid assays, along with other effectors such as the WASH1/strumpellin complex [32] (R. Teasdale, personal communication). The interaction of VPS29 with mammalian SNX proteins is more contentious. To date, we have been unable to detect interactions of VPS29 or the retromer complex with mammalian SNX proteins in similar conditions as those used for TBC1D5 [27], [32]. For example, in experiments shown in Fig. 8A, we over-expressed GFP-VPS29 in HeLa cells in order to enhance potential complex formation, and performed immunoprecipitations with either the core retromer subunit VPS26 or the sorting nexin SNX1. As an initial control we first showed that VPS26 (and hence VPS29-containing retromer) readily binds the protein strumpellin as shown previously [32]. However, no SNX1 is detected in these immunoprecipitations, nor is retromer binding when SNX1 is used as bait, indicating a weak or transient interaction in vivo. The lack of binding is not due to competition by the antibody itself as experiments with epitope-tagged proteins have yielded similar negative results [27], [32]. Although other groups have detected interactions of retromer (both VPS35 and VPS29 subunits) with SNX proteins using ultra-sensitive yeast 2-hybrid assays, or over-expression of all retromer and SNX subunits together [20], [43], [44], there have been no reports of binding between endogenous mammalian proteins.

Bottom Line: VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins.There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case.Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo.

View Article: PubMed Central - PubMed

Affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

ABSTRACT
VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+) and Zn(2+) in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.

Show MeSH
Related in: MedlinePlus