VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins
et al. (2011) VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for
Retromer Interaction with Accessory Proteins. PLoS ONE 6(5): e20420. doi:10.1371/journal.pone.0020420
VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins
James D. Swarbrick 0 1
Daniel J. Shaw 0 1
Sandeep Chhabra 0 1
Rajesh Ghai 0 1
Eugene Valkov 0 1
Suzanne J. Norwood 0 1
Matthew N. J. Seaman 0 1
Brett M. Collins 0 1
Ludger Johannes, Institut Curie, France
0 Current address: Department of Biochemistry, University of Cambridge , Cambridge , United Kingdom
1 1 Monash Institute of Pharmaceutical Sciences, Monash University , Parkville, Victoria , Australia , 2 Institute for Molecular Bioscience , The University of Queensland , St. Lucia, Queensland , Australia , 3 School of Chemistry and Molecular Bioscience, The University of Queensland , St. Lucia, Queensland , Australia , 4 Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge University , Cambridge , United Kingdom
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 Mn2+ and Zn2+ 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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Retromer is a protein complex peripherally associated with
endosomal organelles, and controls trafficking of a number of
critical cargo molecules within tubulovesicular carriers to the trans
Golgi network (TGN) [1,2,3,4]. Biochemical and genetic studies in
yeast and higher eukaryotes have identified two distinct retromer
sub-complexes; a core trimer composed of VPS35-VPS29-VPS26
(VPS: vacuolar protein sorting) and an associated homo or
heterodimer of sorting nexin (SNX) proteins, containing combinations of
SNX1, SNX2, SNX5 and SNX6. The current model postulates
that the core complex is a cargo loading assembly that binds to the
cytoplasmic tails of trafficking receptors such as the cation
independent mannose-6-phosphate receptor (CI-MPR), Wntless,
sortilin and DMT1 via the large VPS35 subunit [5,6,7,8,
9,10,11,12,13,14,15]. The SNX proteins drive the membrane
remodelling required to form the tubulovesicular transport
structures [16,17,18,19], and along with the small GTPase Rab7
may regulate recruitment of retromer to endosomal membranes
through binding to phosphatidylinositol-3-phosphate (PtdIns(3)P)
[18,19,20,21].
The exact roles of the individual subunits of the core retromer
complex remain unclear. The function of the large VPS35 subunit
is perhaps the best defined, as it forms the central scaffold for
assembly with VPS29 and VPS26 [22,23,24,25], it binds directly
to transmembrane cargo molecules [5,7,12], and also associates
with SNX proteins [19,23,26]. It can therefore be thought of as
the primary hub for the spatiotemporal assembly of functional
transport intermediates. The roles of VPS29 and VPS26 are not as
well understood. Each of these proteins is required for the stability
of the core trimer in vivo [22,24,27,28,29], and based on structural
similarity to arrestin molecules it has been suggested that VPS26
may play an ancillary role in recruiting cargo molecules or
accessory proteins [22,29], although there is currently no
experimental evidence for this.
VPS29 is the smallest subunit of retromer and its structure
reveals a striking similarity to Ser/Thr phosphatase enzymes
[24,27,30]. This discovery led to the hypothesis that with VPS35
and VPS26, VPS29 may be the catalytic subunit of a trimeric
phosphatase (core retromer), with some similarity to the PP2A
holoenzyme [2]. Support for this came from the observation that
VPS29 displayed weak phosphatase activity against
phosphorylated (...truncated)