GGA Autoinhibition Revisited

Jacob F. Cramer1, Camilla Gustafsen2,ManjaA. GGAs contain separate functional sites that facilitate their Behrens3, Cristiano L. P. Oliveira3, Jan Skov binding to membranes, accessory proteins, clathrin and speciﬁc sorting motifs in the cytoplasmic domains of Pedersen3, Peder Madsen2, Claus Munck transmembrane cargo proteins (1–5). In GGAs, however, 2,∗ 1,∗ Petersen and Søren S. Thirup the molecular functions relate to a conserved modular organization of a single chain and not to individual subunits 1MIND Centre, Department of Molecular Biology, Aarhus as seen in the AP complexes (reviewed in 6). From the University, Gustav Wieds Vej 10 C, DK-8000 Aarhus C, N-terminus, each GGA contains a VHS domain (Vps27, Denmark Hrs, Stam; ∼140 residues), a 20 residues linker sequence 2MIND Centre, Department of Medical Biochemistry, which is followed by a GAT domain (GGA and TOM; Aarhus University, Ole Worms Alle´ 1170, DK-8000 ∼150 residues), a variable presumably unstructured 80- Aarhus C, Denmark 286 residue hinge region and ﬁnally a C-terminal GAE 3 Department of Chemistry and iNANO Interdisciplinary domain (γ-adaptin ear) (Figure 1A). Whereas binding of Nanoscience Center, Aarhus University, Langelandsgade accessory proteins, clathrin and membrane-associated 140, DK-8000 Aarhus C, Denmark Arf-GTP is accounted for by the GAE domain (7–9), the *Corresponding authors: Søren S. Thirup and Claus hinge regions and the GAT domain (10,11), respectively, Munck Petersen the VHS domain is responsible for binding of the cargo [email protected], [email protected] proteins (12–15).

The cytosolic adaptors GGA1-3 mediate sorting of Although the role(s) of GGAs is far from clarified, it seems transmembrane proteins displaying a C-terminal acidic clear that they contribute, alone or in cooperation with dileucine motif (DXXLL) in their cytosolic domain. GGA1 other adaptors like AP-1 and phosphofurin acidic cluster and GGA3 contain similar but intrinsic motifs that are sorting protein 1 (PACS1), to the sorting and transport believed to serve as autoinhibitory sites activated by the phosphorylation of a serine positioned three residues of receptors between the trans Golgi network (TGN) upstream of the DXXLL motif. In the present study, and endosomal compartments (16). This is underscored we have subjected the widely acknowledged concept by the fact that most of the six identified GGA-binding of GGA1 autoinhibition to a thorough structural and cargo proteins are known to be active in Golgi-endosome functional examination. We find that (i) the intrinsic trafficking. The cargo proteins include the Vps10p- motif of GGA1 is inactive, (ii) only C-terminal DXXLL domain receptors Sortilin (12,14) and SorLA (17,18), motifs constitute active GGA binding sites, (iii) while the cation-independent (13–15) (CI) and the cation- aspartates and phosphorylated serines one or two dependent (13,19) (CD) mannose 6-phosphate receptors positions upstream of the DXXLL motif increase GGA1 (MPRs), the low-density lipoprotein-related protein 3 binding, phosphoserines further upstream have little or (LRP3) (14) and BACE (β-site amyloid precursor protein no influence and (iv) phosphorylation of GGA1 does not cleaving enzyme, β-secretase) (20), and each of these affect its conformation or binding to Sortilin and SorLA. Taken together, our findings seem to refute the functional are characterized by having an ’acidic-cluster dileucine’ significance of GGA autoinhibition in particular and of type motif close to the C-terminus of their cytoplasmic intrinsic GGA binding motifs in general. domain. This motif forms the basis for interaction with the VHS domain of GGAs that have been directed to Key words: acidic cluster dileucine, GGA, VHS domain, the membrane via GAT domain-mediated binding to ARF- phosphorylation, autoinhibition, sortilin, SorLA, x-ray GTP (10). Studies by several groups and implementation of structure various techniques, including yeast two-hybrid and crystal structure analysis, have established that the sequence Received 27 April 2009, revised and accepted for DXXLL (where X could be any residue and M or V could publication 4 November 2009, uncorrected manuscript substitute for L) forms the minimal requirements for GGA published online 10 November 2009, published online 11 binding (19,21–24). However, in addition to the essential December 2009 aspartate (position 0) and dileucine (positions +3and +4), nearby residues both upstream and downstream to the signal are known to modulate the interaction. The Golgi-localized, γ-ear-containing, Arf-binding proteins In the first place, one or more upstream aspartates (GGAs) constitute a family of three (GGA1-3) monomeric and/or phosphorylatable serines are found in all the target adaptor proteins (APs) that are ubiquitously expressed. proteins, and an additional Asp or phosphorylated Ser The GGAs contribute to the formation of coats on the (pS) at position -1 results in a significant increase in cytosolic face of vesicular transport carriers and to the the affinity for VHS-domain binding (25). An effect of D selection and incorporation of specific cargo proteins into or pS at positions -2 and -3, on the other hand, has the carriers. Like the tetraheteromeric AP complexes, not been convincingly demonstrated. Second, previous

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in position -3, the hinge site becomes activated and each of the two adaptors binds their respective VHS domain and undergoes a conformational change (27). Thus, the unphosphorylated adaptor is stated to contain a free VHS domain, which enables it to bind and sort its transmembrane targets, whereas the phosphorylated adaptor is inaccessible to cargo proteins as it engages and inhibits its own VHS domain. Previous reports suggest that the resulting mechanism of regulated autoinhibition may play an important role in the orchestration of AP-1, PACS1 and GGA-mediated Golgi-endosome sorting of cargo such as MPRs and Sortilin (28,29).

In the present study, we have taken a closer look at GGA autoinhibition using peptides and full-length proteins. We have studied the interaction between GGA1, its intrinsic VHS binding site and its transmembrane targets Sortilin and SorLA and have focused on the role of phosphorylatable serines and C-terminal requirements of the binding motifs. Our ﬁndings are incompatible with the existence of a functional intrinsic VHS-binding motif and the concept of GGA1 autoinhibition.

Results

To examine the interaction between GGA1 and established DXXLL motifs, we initially generated a series of peptides derived from the VHS-domain binding C-termini of Sortilin and SorLA, and from the intrinsic ‘autoinhibitory’ site in the hinge region of GGA1 itself. The peptides include unmodified sequences (designated So, Sa and Hi, respectively) as well as phosphorylated (SoP, SaP, HiP), Figure 1: Schematic representation of GGA structure and point mutated and extended peptides (Figure 1B). DXXLL peptides. A) The structure of GGA1 with proteins that bind to each domain indicated by arrows and the autoinhibitory For reference, we first determined the structures of the sequences of GGA1 and GGA3 shown together with the DXXLL unmodified peptides in complex with the GGA1-VHS motif of Sortilin, SorLA, CI- and CD-MPR, LRP3, LRP9 and BACE. domain. The structures of VHS:So and VHS:Sa were The three critical residues in the DXXLL motif are marked by grey determined at 2.3 and 1.7 A,˚ respectively. As can be and serine residues that are potential targets for phosphorylation seen (Figure 2A–D), the seven C-terminal residues of are marked by red. B) Sequence of DXXLL peptides from GGA1, So and the eight C-terminal residues of Sa are clearly Sortilin, SorLA and LRP9 used in this study. defined in the electron density and firmly bound in the depression between helices α6andα8 of the VHS domain. findings have strongly indicated that whereas one or Notably, both peptides are anchored by tight positioning two C-terminal residues (positions +5and+6) may of the three key residues, i.e. the aspartate at position 0 help to stabilize the interaction, additional C-terminal and the ‘dileucine’ at position 3-4. Thus, the sites in residues may actually abrogate the signal and abolish Sortilin and SorLA bind GGA1 in accordance with previous binding (19,22). In other words, the GGA-binding motif is observations on similar complexes involving the signal β only active when found at the extreme C-terminus of the peptides of the MPRs and the -secretase BACE (22–24). cytoplasmic domain. A more detailed description of the two structures is found as supplementary material. In light of this, it is obviously surprising that the GGAs themselves have been reported to contain functional Only phosphoserines immediately upstream to the intrinsic DXXLL sites for VHS-domain binding (Figure 1A; DXXLL motif contribute to VHS-domain complex 26). The sites are located in the hinge region of GGA1 formation and 3, but not in GGA2, and both GGAs are supposed Each of the three peptides So, Sa and Hi contains to exhibit regulated autoinhibition by binding their own a single upstream phosphorylatable serine found at VHS domain (26). The native proteins show no intrinsic positions -1 (So), -2 (Sa) or -3 (Hi). To investigate if binding but following phosphorylation of a serine residue and how phosphorylation of these serines could facilitate

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Figure 2: Interaction between the GGA1-VHS domain and DXXLL peptides from Sortilin and SorLA. A and B) Overall architecture of the VHS:So (Sortilin) and VHS:Sa (SorLA) complex, respectively, with the VHS domain shown by surface representation and the bound DXXLL peptide molecule as a ’ball-and-stick’ model coloured by atom type (nitrogen, blue; carbon, yellow; oxygen, red). The two helices, α6andα8, are shown together with side chains that form the binding pocket at the VHS surface (grey, transparent). C and D) Molecular details in top view of So and Sa binding on the VHS domain, respectively. The interacting part of the So and Sa peptide is shown in centre (ball-and-stick model) and superimposed a simulated annealing (SA) omit difference electron density map, contoured at 2.8 and 2.7 σ, respectively. Residues from the helices 6 and 8 of the VHS domain involved in the interactions with the peptide is shown (nitrogen, blue; carbon, orange; oxygen, red; sulphur, yellow) and labelled with single letters. Hydrogen bonds formed between the peptide and the VHS domain are coloured black and interactions with the key residues at positions 0, 3 and 4 of the peptides are shown in enlarged views.

GGA binding, we next determined the structures of the involving a positively charged patch formed at the VHS- phosphorylated peptides SoP, SaP and HiP in complex domain surface by the side chains of Lys87, Arg89 with the VHS domain. The crystals of these complexes and Lys131. The pS(-1) of Sortilin interacts with Lys131 were obtained under the same conditions and on the through a bridging water molecule and with Lys87 by same form as crystals containing unmodified peptides a weak electrostatic interaction (Figure 3A). Interestingly, and the derived structures were refined to high resolution the pS(-1) of the practically identical signal peptide of (Tables S1 and S2). It appears (Figure 3A–D) that each CI-MPR was found to interact chiefly with Arg89 (25) peptide is found at the anticipated site between α6andα8 (Figure 3D). This and the present finding no doubt reflect of the VHS domain anchored by Asp(0) and by the dileucine that pS(-1) is ‘flexible’ and capable of engaging the VHS motif at position 3-4. The central and C-terminal parts of domain by alternative interactions. The pS(-2) of SorLA, on the phosphopeptides are well ordered and superimpose the other hand, seems to have only one option. It needs nicely with their unmodified counterparts (Figures 3E to ‘reach back’ to the electrostatic patch and this implies a and S1). Also, the phosphoserines of SoP and SaP are sharp turn in the peptide, which is stabilized by interactions seen to contribute to peptide binding via interactions between the backbone carbonyl of pS(-2) and Lys87, and

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(or Asp) at positions -1 or -2 is optimally placed for interaction with the conﬁned basic ‘patch’ (Lys87, Arg89 and Lys131) (Figure 3E). In contrast, negatively charged residues further upstream are removed from the vicinity of the basic cluster and left with no or less favourable alternative sites (Figure 3E). It follows that upstream acidic side chains must be placed within 1 or 2 positions from the main DXXLL motif to enhance peptide binding.

This is evidently highly surprising as phosphorylation of this exact serine (S355 in wt GGA1) was reported to trigger intrinsic VHS-domain binding (autoinhibition) (26) and thereby to prevent GGA1 and 3 from targeting their cargo proteins.

Affinity of signal peptides for the VHS domain To further substantiate these findings, we next examined the binding of wild-type (wt), mutant and phosphorylated signal peptides to the VHS domain using isothermal titration calorimetry (ITC). The results are displayed in Figure 4, and show that the wt peptides So and Hi bound with similar Kds, whereas Sa [which, notably, contains an Asp(-1)] exhibited a ∼10-fold higher affinity. Moreover, it appears that phosphorylation of the Sortilin and SorLA peptides was accompanied by a significant increase in binding activity. Thus, the presence of a pS(-1) in SoP and a pS(-2) in SaP increased the affinity by ∼18-fold and ∼6-fold, respectively. In contrast to this, the Kd of the hinge region peptide was altered by about 2.6-fold upon phosphorylation of its Ser(-3), while substitution of Ser with Asp or Glu had no effect at all.

Figure 3: Structures of the GGA1-VHS domain in complex with phosphorylated DXXLL peptides. The phosphopeptide–VHS complexes are shown for comparison in the same view. The overall architecture of the complex is shown at the left and an enlarged top view of the phosphoserine binding site is given to the right. All peptides (CPK bonds) are bound between helices 6 and 8 of the VHS domain with the phosphoserine residue placed for interaction with K87, R89 and K131 from the VHS domain (stick model, carbon: light yellow). The interaction near the phosphoserine is shown by dashed lines and the electron density for this part of the phosphopeptide is shown as a (Fo − Fc)SAomit map (phosphopeptide omitted, map contoured at 2.3 σ). Peptide between Asp(-1) and Arg89. The resulting conformation residues are labelled by single letters and their relative position then provides the basis for electrostatic binding between with triangles. A) Structure of the phosphorylated Sortilin peptide the phosphoserine and Lys131 (Figure 3B). (SoP) bound to the VHS domain of GGA1. B) Phosphorylated SorLA peptide (SaP) bound to the VHS domain of GGA1. C) Phos- In contrast to the above, the pS(-3) of the hinge region phorylated hinge peptide from GGA1 (HiP) bound to the VHS peptide does not appear to participate in peptide binding domain of GGA1, with the phosphoserine residue at position -3 (Figure 3C). The HiP peptide is placed in the binding pocket not visible in the electron density due to its ﬂexibility. D) Struc- similar to SoP and SaP, but shows no electron density ture of the phosphorylated CI-MPR peptide bound to the VHS corresponding to the upstream phosphoserine. Thus, domain of GGA3 (25) (PDB: 1LF8). Residues of the VHS domain Leu(-2) is the most N-terminal residue visible, strongly are numbered according to the GGA1. E) The phosphopeptides SoP, SaP, HiP and CI-MPR-P are superimposed and shown at the indicating that the pS(-3) does not facilitate binding to the VHS surface with the phosphoserine site outlined (at left). The VHS domain. The most likely explanation for this is the VHS surface is coloured according to its electrostatic potential displacement relative to the anchoring Asp(0) in the main (blue, positive; red, negative; scale from −20 to +20 kT/e) and binding site. When examining the electrostatic surface the phosphoserine site is shown in an enlarged surface view at of the VHS domain, it is clear that a phosphoserine the right.

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Figure 4: Binding of phosphorylated and unphosphorylated Sortilin, SorLA and hinge peptides to the VHS domain of GGA1. A) ITC measuring the binding of the phosphorylated SoP and unphosphorylated So peptide to the VHS domain of GGA1. The inset shows the differential heat released when 0.81 mM So was injected into 35 μM GGA1-VHS. The trace is shown after subtraction of data from the injection of peptide into a buffer blank. Binding constants are listed in (D). B) ITC of the SaP and Sa. Insert: Differential heat released when 0.24 mM Sa is injected into 11 μM GGA1-VHS. C) ITC of hinge peptides: Unphosphorylated (Hi, filled squares), phosphorylated (HiP pS355, filled circles) and phosphorylation mimic (Hi S355D, empty circles) and (Hi S355E, filled triangles). Insert: Differential heat released when 0.24 mM Hi is injected into 6 μM GGA1-VHS. D) Dissociation constant (Kd) and stoichiometry for the different peptide-VHS interactions expressed as mean ± standard deviation

GGA1 binding to cargo proteins is unaffected by demonstrate that all GGA constructs bound to both recep- phosphorylation of Ser355 tors. Similar results, suggesting much the same binding Surprisingly, the S355D mutation did not mimick phos- of all constructs, were obtained when the cytoplasmic phorylation in the peptide binding assay, although this has domains of Sortilin and the CI-MPR were employed for previously been proposed to be the case (26). To examine pull down of GGA1 constructs (wt, S355D and S355A) if such an effect was present in the full-length protein, we stably expressed in HEK293 cells (Figure 5E). Similarly, next performed pull-down experiments using full-length testing of full-length constructs in a yeast two-hybrid GGA1 and lysates of cell transfectants expressing its system showed no signiﬁcant difference between the cargo receptors Sortilin and SorLA. Wt GGA1 or mutant reactivity of wt GGA1 and the two mutants (Figure 6A). constructs, i.e. S355D (mimicking permanent phosphory- Taken together, the above is in agreement with our anal- lation) or S355A (excluding phosphorylation), were added ysis of the VHS-domain:peptide complex structures and to the lysates. Following incubation, pull down was the data on signal peptide binding, but clearly contrary to then performed using antireceptor antibodies on samples reports stating that phosphorylation of S355 (or a S355D containing glutathione S-transferase (GST)-tagged GGA1 mutation) elicits GGA1 autoinhibition (26,27). (N-terminal), and Talon beads on samples containing His6- tagged GGA1 (C-terminal). Finally, binding (coprecipitation) We conclude that upstream phosphoserines and acidic between GGA1 and SorLA/Sortilin was assessed by west- residues beyond the (-2) position of the D(0)XXL(3)L(4) ern blotting. The results are depicted in Figure 5A–D, and motif have little or no inﬂuence on VHS-domain binding.

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Figure 6: Yeast two-hybrid analysis of the interaction between GGA1 and DXXLL motifs. A) The cytoplasmic domain of Sortilin, with and without a C-terminal myc-tag, was tested in combination with wt GGA1 and with mutant GGA1 constructs. In the mutants, the phosphorylatable serine Figure 5: Coprecipitation of GGA1 constructs and receptor of the assumed autoinhibitory site was substituted with either tails. Full-length GGA1 constructs were generated as fusion an aspartate (S355D; mimicking phosphorylation) or an alanine proteins containing GST-tags (N-terminal) or 6His-tags (C- (S355A; excluding phosphorylation). Controls for autoreactivity terminal). For precipitation, puriﬁed GGA1 proteins were added were included as indicated. B) The full-length CI-MPR-wt and to lysates of cells expressing full-length wt Sortilin, full-length wt the myc-tagged variant (CI-MPR-myc) were tested along with SorLA or C-terminally truncated (tr) SorLA not containing the GGA- the LRP9-wt and the C-terminally truncated mutant (LRP9-del). binding motif. Pull down was performed using Talon beads (B Controls for autoreactivity were included as indicated. and C) or Sepharose beads coated with antireceptor antibodies (A and D) and precipitates were subsequently analysed by western blotting using antireceptor antibodies (B and C) and anti-GST Ig (A It follows that the Ser(-3) in the signal sites of the GGA1 and D), respectively. S/A and S/D indicate GGA1 mutants in which and GGA3 hinge regions is not in a position to regulate the phosphorylable serine of the assumed autoinhibitory site has intrinsic VHS-domain binding and autoinhibition. been substituted with either alanine or aspartate (mimicking phosphorylation). Pull down with the GAE and VHS domains of GGA1 were included as additional negative and positive controls. C-terminal requirements for GGA binding In addition, lysates of HEK293 cells expressing full-length GGA1 In addition to the described upstream acidic residues, wt, S/A or S/D mutants and untransfected cells were incubated residues adjacent to the C-terminus of the main motif with GST fused to the cytoplasmic domain (cd) of Sortilin (So), are also crucial for binding. As shown in Figure 7, the the 21 terminal amino acids of CI-MPR (MPR) or GST alone (-). converging C-termini of the α6andα8 helices of the VHS Pull down was performed using glutathione beads (E). GGA1-myc domain, and the loop between α6andα7, form a tight proteins were detected with anti-myc. E) The upper panel shows pocket that harbours the C-terminus of the So, Sa and the expression level of GGA1 in the lysate from transfected and untransfected cell lines (1% of total input volume). The lower HiP peptides. The pocket is lined with polar residues, panel shows precipitated GGA1 proteins. dimensioned to ﬂank two residues and it constitutes a basis for interactions with residues at positions 5 and 6

264 Traffic 2010; 11: 259–273 GGA Revisited in the peptides. In Sa and Hi, the terminal carboxy group of binding activity (Figure 7E). Also in the yeast two- (Ala6 and Leu6, respectively) is firmly bound by hydrogen hybrid setup, both the Sortilin and the CI-MPR tails were bonds to Ser99 and Gln142, and in So, which is one residue completely unreactive upon addition of C-terminal myc- shorter, the terminal carboxy group (Glu5) is still within tags (Figure 6A,B). LRP9 was recently reported to contain distance to interact with Tyr102 and Gln142. This explains both a C-terminal and an internal GGA-binding site (30). why lack of downstream residues (positions 5 and 6) filling To assess the function of these two sites, wt LRP9 the pocket may weaken peptide binding (22). On the other cytoplasmic domain and a truncated version (missing hand, they also suggest that additional C-terminal residues the C-terminal GGA site) were tested in the yeast two- may seriously hamper binding as the confined space of hybrid setup in parallel with the CI-MPR for binding of the pocket seems unable to accommodate more than full-length GGA1 (Figure 6B). Compared to the CI-MPR two residues. Thus, a longer downstream segment would cytoplasmic domain, the wt LRP9 cytoplasmic domain require a significant displacement of the loop between α6 reacted well (in terms of beta-galactosidase activity), while and α7 of the VHS domain or a sharp bend in the peptide, the truncated LRP9 cytoplasmic domain produced no both of which are unlikely. To clarify this, we therefore induction of beta-galactosidase activity. In agreement, examined the binding of the Hi and the So peptides ITC measurements demonstrated that truncation left before and after they had been C-terminally elongated. LRP9 with almost no affinity for GGA1. Once again, this As apparent from Figure 7, the addition of merely three gives strong evidence that only C-terminal sites can be alanines to the C-terminus of the So peptide resulted in an considered functional. ∼18-fold downregulation of its affinity for the VHS domain and a C-terminal myc-tag yielded additional loss of affinity. It can be concluded that DXXLL motifs with a spacing Likewise, elongation of the Hi peptide with eight residues, of four or more residues to the C-terminus, be it natural i.e. the sequence that normally follows the peptide in intrinsic sequence or tags, do not constitute functional wt GGA1, was accompanied by almost complete loss targets for GGA binding in vivo (Figure 7F).

Figure 7: Requirements for C-terminus interaction. A–C) C- terminus interaction of Sa, So and HiP with the VHS of GGA1, respectively. The VHS domain of GGA1 is shown as a ribbon diagram with interacting side chains and the peptide residues as ’ball-and-stick’ model coloured by atom type (nitrogen, blue; oxygen, red; carbon, individually). The C-terminal part of the bound peptide molecules is superimposed on a transparent surface (Sa, teal; So, yellow; HiP, purple) and hydrogen bonds formed to interacting side chains coloured black. D) In top, ITC measuring the afﬁnities of the C-terminal-extended Sortilin (SoA: extended with three alanines; SoMYC: extended with a myc-tag) and hinge (HiX: extended with eight downstream residues) for the VHS domain of GGA1. Insert: Differential heat released when 0.45 mM HiX was injected into 10 μM GGA1-VHS. In bottom, ITC measuring the afﬁnities of the LRP9wt and LRP9del for the VHS domain of GGA1. Insert: Differential heat released when 0.024 mM LRP9wt was injected into 0.8 μM GGA1-VHS. E)Kd and n (stoichiometry) for the peptide-VHS interactions expressed as mean ± SD. F) Scheme showing the C-terminal requirements for peptide binding at the end of the DXXLL pocket.

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Figure 8: Gel filtration of GGA1. A) GGA1 wt and mutants were gel filtrated at the varying concentrations: c, c/2, c/4 and c/20 using a Superpose 6/100 column. In chromatograms, the proteins are shown as wt (solid), S355A (filled circles), S355D (filled triangles) and GGA1-short (dotted). Insert shows the standard curve used for determination of Stoke’s radius. B) Stoke’s radius determined from gel filtration at high (c) and low (c/20) concentration. The molecular radius was determined for the major species at both concentrations, corresponding to dimer and monomer, respectively. C) Identification of the complex between the Sortilin cytoplasmic tail and the GGA1 proteins. Gel filtration (Superose 6 10/300) is shown in top of the Sortilin tail (squares) and the GGA1 proteins as either monomer (c/20) or dimer (c), respectively. In bottom, gel filtration chromatography is shown of GGA1 proteins premixed with the Sortilin tail at a 1:4 molar ratio. Proteins are given as in (A) and SDS–PAGE is shown of all peak fractions.

Solution structure of wt GGA1 and mutants Analytical gel filtration was performed on GGA1 wt, Using analytical gel filtration, intrinsic fluorescence, small S355D, S355A and on the truncated GGA1 construct, angle X-ray scattering (SAXS) and dynamic light scattering which completely lacks the intrinsic DXXLL motif (i.e. (DLS), we next examined if mutations in the autoinhibitory residues 274-362). At 7 mg/mL ([c]), all four proteins site of GGA1 had any effect on the overall conformation eluted as single peaks and their estimated Stoke’s of full-length GGA1. radii were essentially identical (∼60 A,˚ Figure 8A,B),

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Figure 9: SAXS and DLS analysis of GGA1. A) Comparison of scattering data of GGA1 wt (black), GGA1-S355D (blue) and GGA1-S355A (red) at the concentration of c/2 (right) and c/4 (left). B) The apparent molecular mass, radius of gyration and maximal dimensions of the GGA1 proteins obtained by SAXS. C) DLS mea- surement of GGA1 proteins at the concentration c/2, coloured as in (A). Stoke’s radius is indicated for the dominating species. suggesting a similar intramolecular arrangement of and both point mutations of Ser355 appeared to have an domains. Accordingly, the four constructs showed effect on the monomer/dimer equilibrium. This suggests minor difference in peak intensity, as determined that, in addition to the previously described interaction by fluorescence spectra, but no difference in peak between a hydrophobic motif in the hinge region and the emission wavelength (Figure S3A,B). To detect possible GAE domain (31), a direct interaction between the GAE oligomerization, the analysis was repeated at lower dimer and the hinge region may exist. concentrations; [c]/2, [c]/4 and [c]/20 (see Materials and Methods). This procedure did not significantly change SAXS measurements on GGA1 wt, S355A and S355D the fluorescence of the proteins but altered their elution (Figure 9A) were carried out at concentrations [c]/2 and time. At [c]/20, the estimated Stoke’s radius had shifted [c]/4. The two sets of scattering curves show that the from ∼60 to ∼40 A,˚ which would agree with a shift from wt GGA1 and the S355A mutant are very similar at [c]/2 dimers to monomers (Figure 8A). However, as shown in and almost identical at [c]/4 (reduced chi-square χ2 = 2.15 Figure 8C, both forms of all four proteins were capable of and 1.13, respectively). In contrast, scattering from the interacting with the Sortilin cytoplasmic tail, implying that S355D mutant differs markedly at both concentrations, the dimer formation does not block access to the DXXLL- in agreement with the distribution of ∼60 and ∼40 A˚ binding site in the VHS domain. The deletion mutation forms obtained by gel filtration, and the molecular masses

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Figure 10: Solution structure of GGA1. A) Rigid body modelling of the GGA1 dimer (wt) at concentration c/2. B) Model of GGA1 dimer in solution. The average of the 10 runs is shown in semitransparent beads and the best model is superimposed on the average. The three known domains are shown: the VHS domain (yellow, PDB ID: 1JWF), the GAT domain (blue, PDB ID: 1O3X) and the GAE ◦ (pale green/green, PDB ID: 2DWY). The model is rotated 90 . C) The calculated Stoke’s radii from the SAXS rigid body model and DLS measurements. derived from the scattering further support the notion interaction between the structural domains is scarce and that the samples constitute a mixture of monomers and the domains of GGA1 are primarily held together by limited dimers (Figure 9B) interactions mediated by the linker regions (Figure 10B). In the model, the distance between the C-terminal of the DLS measurements (at [c]/2) also confirmed the presence VHS domain and any part of the hinge region is larger of two protein species with Stoke’s radii of about 60 than 40 A,˚ making an interaction between the intrinsic and 40 A˚ and the shift in equilibrium between the two DXXLL motif and the VHS domain highly unlikely. The introduced by the S355D mutation (Figure 9C). rigid body fitting was repeated using the other known dimer conformation of GAE as constraint (8). The quality of the fit with the experimental data was essentially the Modelling SAXS data same (Figure S5) and the overall conclusion remained the A model of the GGA1 dimer was created by rigid body same. In particular, the distance between the C-terminal fitting of the known structures of the VHS, GAT and of the VHS domain and the hinge region was again found GAE domains to the SAXS scattering curve obtained at to be larger than 40 A.˚ [c]/2, and using the additional constraint that the GAE domain forms a tight dimer (see Figure S3 for details), but not including the constraint that residues 381-387 Discussion of GGA1 binds to the GAE domain (31). A good fit was obtained for predicted versus observed scattering data at After the first demonstration that GGAs target cytosolic concentration [c]/2 (Figure 10A). The overall dimension of receptor domains for sorting, acidic-cluster dileucine the resulting model fully meets our expectations as the motifs of the DXXLL type were quickly identified as the calculated dimer Stoke’s radius (∼60 A)˚ is in very good functional sites for GGA-VHS domain binding (12–15). agreement with the DLS measurements (Figure 10C). In Initial studies further indicated that phosphoserine or the model, the six domains of the wt GGA1 dimer are aspartate residues immediately upstream to the main forming an elongated structure, where only the GAE signal serve to enhance binding and, notably, that domain is responsible for dimer formation. Within each functional signals are situated close to the C-terminus of monomer, the VHS domain and the GAE domain are cargo proteins (19,22,25). In agreement, the DXXLL motif placed at opposite sides of the GAT domain. Direct of all primary targets for GGA interaction, i.e. Sortilin,

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SorLA, the MPRs, LRP3 and the β-secretase (BACE), affinity of Sortilin, and of S(-2) enhanced that of SorLA by is separated from the C-terminus by only one or two about 6-fold. Phosphorylation of the S(-3) (or substitution residues. More recently, however, the requirement for a with Asp/Glu), on the other hand, had little or no effect on C-terminal localization has been seriously questioned. the binding of the internal GGA1 peptide. Based on direct and indirect evidence, a number of studies have reported the identification of GGA-binding internal In agreement with this, full-length proteins were not DXXLL motifs (30,32). Most importantly, the GGAs affected by phosphorylation. Neither yeast two-hybrid themselves contain internal DXXLL-type sites and both nor pull-down analysis showed any difference between GGA1 and -3 have been reported to exhibit autoinhibition the binding activity of full-length wt GGA1 and that of by binding their own VHS domains to such sites (26). GGA1 mutants, which were either mimicking permanent phosphorylation (S/D) or resistant to phosphorylation It is obvious that the verification or dismissal of these (S/A). Previous investigation of CD-MPR interaction with findings is highly significant to our conceptual under- GGA1 stay in line with this, as a negatively charged standing of protein sorting in general and of regulated residue at position (-2) showed to be critical for binding, GGA function in particular. In the present study, we have whereas phosphorylation further upstream (-4) was therefore employed a broad range of techniques to clar- without importance (33). ify some basic requirements for GGA interaction with DXXLL motifs. To that end, we have re-examined previous Based on the above, we can only conclude that findings with particular reference to the reported (casein phosphoserines located beyond upstream position (-2) kinase 2) phosphoregulated autoinhibition of GGA1 (26). of the DXXLL motif have little or no bearing on VHS-domain binding. It follows that phosphorylation of Ser355 in GGA1 (and -3) is very unlikely to serve The influence of upstream phosphoserines as an on/off switch in GGA autoinhibition. The latter The phosphorylation of Ser355 positioned at (-3) upstream conclusion is further substantiated by results obtained to the internal DXXLL site in GGA1 (and in GGA3) by gel filtration. A ‘functional’ phosphoserine would be is essential to the whole concept of autoinhibition. In expected to induce a conformational change in GGA1, but the phosphorylated state, the serine is said to elicit chromatography displayed uniform elution profiles and GGA1 autoinhibition by facilitating interaction between similar Stoke’s radii for the wt and mutant (S/D or S/A) full- the DXXLL site and the VHS domain of the same length GGA1. Likewise, intrinsic tryptophan fluorescence GGA1 molecule (26). Obviously, this is only possible if gave no evidence of a conformational change upon the pS(-3) (compared to an unphosphorylated S or A at phosphorylation. Interestingly, the SAXS data suggest the same position) causes a considerable increase in that GGA1 monomers interact via their GAE domain the affinity of the internal site for VHS-domain binding. to form dimers (Stoke’s radius ∼60 A),˚ which at low It is an established fact that phosphoserines upstream concentrations tend to dissociate into monomers (Stoke’s to the DXXLL binding site enhance binding, but ‘how radius ∼40 A).˚ It appears that the GGA1 S/D mutant far upstream’ is unclear. To examine this, we have dissociates more readily than the GGA1 wt and this used DXXLL peptides derived from GGA1, SorLA and might explain previous observations of a Sp (or D)-induced Sortilin carrying phosphorylatable serines at positions change into an ‘autoinhibited’ molecule with a smaller (-3), (-2) and (-1), respectively. In the unphosphorylated Stoke’s radius (27). state, these peptides all bind in accordance with the previous structure analysis of VHS domains in complex with corresponding peptides of the CI-MPR (24). Our C-terminal requirements present structure analysis of complexes incorporating Another key question is then whether internal DXXLL phosphorylated peptides further shows that Sp’s at motifs are in fact functional VHS-domain binding signals. positions (-1) and (-2) contribute to binding via electrostatic Early results suggested that motifs with a spacing of interaction with a patch of basic residues on the surface more than two or three residues between the dileucine of the VHS domain. At position (-1), the phosphoserine is and the receptor C-terminus were ineffective (19,22). Yet, in close contact and readily interacts with the electrostatic this view has been seriously challenged, not only by patch, and may even [as determined by our findings in the reported GGA autoinhibition (26) but also by recent Sortilin and previous findings in the identical CI-MPR reports of an internal GGA binding site in LRP9 (30,32). peptide (25)] engage in alternative interactions. At position In the present study, we have addressed all the latter (-2), the Sp seems more restricted but is still within reach of reports and find no evidence that support the existence the electrostatic patch and able to contribute visibly to the of functional internal binding sites. In the first place, it VHS:SorLA-peptide complex formation. In contrast, the appears that the structure of the DXXLL-peptide:VHS- Sp of the internal GGA1 peptide is not seen in the crystal domain complex can incorporate no more than two structure, strongly suggesting that at position (-3) it is to C-terminal residues (following the dileucine). Incorporation far away from the electrostatic patch and unable to engage of longer sequences would require an unlikely dislocation in binding. Our ITC analysis of peptide binding in solution of the loop between the α6andα8 helices of the VHS fully supports these findings. Thus, phosphorylation of domain or an equally unlikely kink in the peptide. Second, S(-1) caused a remarkable ∼20-fold increase in the binding C-terminal elongation with six residues of the wt GGA1

Traffic 2010; 11: 259–273 269 Cramer et al. sequence almost abolished binding of the autoinhibitory 21 amino acids of CI-MPR were expressed as GST-fusion proteins using the peptide to the VHS domain. On top of this, neither pGEX4T-1 vector (GE Healthcare). 6His-tagged proteins were purified from the truncated cytoplasmic domain of LRP9 (without the E. coli lysates on Talon beads (Invitrogen) or Ni-loaded HiTrap chelating HP columns (GE Healthcare) and proteins with GST-tags were purified using C-terminal DXXLL-site) nor the Sortilin cytoplasmic domain glutathione-Sepharose beads (GE Healthcare). For detailed descriptions, carrying a C-terminal myc-tag showed any interaction see Supporting Information. HEK293 cell lines expressing SorLA and with the GGA1-VHS domain in the yeast two-hybrid Sortilin were established as earlier described (18,37). C-terminally myc- analysis. We conclude that internal DXXLL motifs are non- tagged GGA1 (wt, S355A and S355D) proteins were cloned in site A of a functional in terms of VHS-domain binding. In general, this pIRES vector (Clontech) with a Zeocin resistance gene cloned in site B, ® means that GGA1 does not target or sort transmembrane and expressed in HEK293 after transfection with FuGENE transfection μ proteins that carry only internal acidic-cluster dileucine reagent (Roche). Stably transfected clones were selected using 300 g/mL Zeocin (Invitrogen). sites or C-terminal tags (34,35). More specifically, it also establishes that full-length GGA1 (and in all probability GGA3) does not contain a functional VHS-domain binding Crystallization and structure determination autoinhibitory site. DXXLL peptides from Sortilin and SorLA (So, SoP, Sa and SaP) and the autoinhibition signal of GGA1 (HiP) were all dissolved in 1 mM Tris–HCl, pH 8.0 and complexed with GGA1-VHS at a peptide-to-protein ratio of It has been argued that the weaker affinity for the 4:1. The different complexes were in general crystallized near the same intrinsic DXXLL motif was compensated by a locally high conditions: 14–22% (w/v) polyethylene glycol (PEG) 5000 MME, 0.2–0.4 M concentration of the motif. Given the distance between NH4I, 0.3 M 1,6-hexanediol and 0.1 M MES-NaOH pH 6.0–6.5. X-ray the VHS domain and the hinge region (>40 A)˚ and the Kd diffraction data were collected using synchrotron radiation on station of the HiX peptide (<200 μM), one can estimate that only BW7B at EMBL/DESY Hamburg, station X06 at SLS Zurich ¨ and stations half the sites of VHS would be occupied by the intrinsic ID14-2, ID14-3 and ID23-1 at ESRF Grenoble, see Table S1. For structure DXXLL motif. This is certainly not enough to constitute an determination, the structure of VHS domain from GGA1 (PDB entry: 1JWG) was used as a starting model and further refined using CNS (version on/off switch in GGA1 function. 1.2) (38) and REFMAC5/CCP4 (39) with the crystallographic statistics listed in Table S2. In all complexes, the peptide could manually be build into the In summary, we find that (i) functional GGA binding model after a few rounds of refinement guided by difference Fourier is limited to DXXLL sites that found no more than (Fo − Fc) and simulated annealing omit maps calculated in Phenix (40), 1-3 residues from the cytoplasmic C-terminus of cargo clearly defining the peptide orientation and conformation. All structures proteins, (ii) only phosphoserines positioned within two contained two molecules of the complex in an asymmetric unit (protein A complexed with peptide C, and protein B with peptide D). The final residues upstream to the main site markedly enhance model quality was analysed using PROCHECK (41) and MolProbity (42) GGA binding, (iii) GGA1 (and most likely GGA3) is not and figures were prepared with Pymol (http://www.pymol.org). Details are a subject to autoinhibition and does not undergo major given in Supporting Information. phosphoserine-induced conformational change.

The conclusions are based on several lines of evidence Isothermal titration calorimetry Binding of peptides to the VHS domain of GGA1 was measured at 308 K and strongly suggest that recent reports incorporating by ITC using a Microcal VP-ITC MicroCalorimeter (Microcal Inc.). GGA1- the concept of GGA autoinhibition, the binding of GGAs VHS was extensively dialysed against 100 mM NaCl, 50 mM Tris–HCl pH to internal DXXLL motifs and the use of (GGA-) cargo 7.5 buffer and following concentrated to 0.05 mM before use. Peptides proteins with C-terminal tags, should be revaluated. were dissolved in the same buffer and to ensure accurate measurements, concentration of protein and peptides were determined by total amino acids analysis as described previously (43). In a typical ITC experiment, Materials and Methods 1.45 mL of 0.025 mM GGA1-VHS was titrated with 0.1 mM peptide in 30 steps of 10 μL. The time between injections was set to 2 min and the Peptides syringe mixing speed was set at 300 rpm. Heat evolving from dilution was Chemically synthesized peptides were provided by Biomol Research measured by injecting the ligand into the buffer. This heat was subtracted Laboratories Inc. and Caslo Laboratory, puriﬁed by high-performance from the heat of reaction to obtain effective heat of binding. The binding liquid chromatography (HPLC) to a purity of 95% or greater and stoichiometry, equilibrium dissociation constant (Kd), enthalpy and entropy validated by Matrix assisted laser Desorption Ionisation Mass Spectrometry were determined using the calorimetric analysis program ORIGIN ver. 7.03 (MALDI-MS). (Microcal Inc.).

Generation of proteins Pull-down experiments The DNA fragment corresponding to the VHS domain of human GGA1 HEK293 cells stably expressing Sortilin, SorLA or GGA1 were lysed on (residues 1-147), was cloned into pGEX4T-1 plasmid (GE Healthcare), ice in 1% Triton-X-100 buffer containing 150 mM NaCl, 2 mM MgCl2, expressed in Escherichia coli BL21 RIL cells and puriﬁed as a GST-fusion 2mM CaCl2,10mM HEPES (pH 7.4) and supplemented with proteinase protein. Before crystallization, the VHS-domain was released by thrombin inhibitors (CompleteMini, Roche). Debris was removed by pelleting and cleavage and gel ﬁltrated using a Superdex 75 column. The GGA1 (Genbank 100 μL of the supernatant was supplemented with 900 μL buffer (without acc. no. NM 013365), GGA1-short (Genbank acc. no. NM 001001560, detergent) prior to addition of 20 μg of GST- or 6His-tagged GGA1 proteins lacking residues 274-362) and wt mutants (S355A and S355D) were either (wt, mutants S/D and S/A, GAE domain and VHS domain), or 10 μg expressed with C-terminal 6His-tags using the pET11a vector (Novagen) or of GST fused to cytoplasmic domains of Sortilin or CI-MPR. Following ◦ with N-terminal GST-tags using the pGEX4T-1 plasmid (GE Healthcare). The overnight incubation at 4 C, pull down on samples containing GST- GAE and VHS domains of GGA1 were expressed with N-terminal 6His-tags fusion proteins was performed using GammaBind G-Sepharose beads using the pT7-PL vector, a derivative of the plasmid pRK172 containing a T7 coated with antibodies directed against the luminal domain of either promoter (36). The Sortilin full-length cytoplasmic domain and the terminal SorLA or Sortilin (44), or by Glutathione Sepharose™4B (GE Healthcare).

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Precipitation from samples containing 6His-tagged proteins was done minimum had been reached and the most probable model and the averaged in 20 mM imidazole using Talon metal-affinity resin beads (Clontech). structure were extracted using program DAMAVER (48) (Figure S3F). The Washed beads were finally resuspended in SDS sample buffer (2 mM hydrodynamic (Stoke’s) radius was calculated for the model dimer and dithioerythritol) and precipitated proteins were analysed by reducing the constituting monomer by use of HYDROPRO (49). The final models SDS–PAGE and immunoblotting against SorLA, Sortilin, GST (anti-GST presented for the SAXS results were made using program MOLMOL (50). 27-4577-01; GE Healthcare) or myc (monoclonal anti-myc 9E10). Similar calculations were performed for another known conformation of the dimerization centre and the results are shown in Supporting Information (Figure S5). Yeast two-hybrid Yeast two-hybrid analysis was conducted using the Matchmaker LexA system (Clontech). The full-length cytoplasmic domains of Sortilin and Dynamic light scattering CI-MPR (wt and wt with C-terminal myc tag) were cloned into the pLexA DLS was performed on a light scattering instrument from ALV. ◦ vector (Clontech). In addition, the C-terminal 65 amino acids from the Measurements were performed at 90 at a protein concentration of mouse LRP9 cytoplasmic domain and a mutant lacking 6 C-terminal amino 3.5 mg/mL. The software delivered with the system was used for deriving acids were cloned into pLexA. GGA1 full-length constructs (wt, S355A and the Stoke’s radius using cumulant analysis. S355D) were cloned into pBAD42 and cotransformed with pLexA plasmids and yeast cells were plated for plasmid selection. Resulting colonies were replica plated on X-gal/induction plates together with controls for Accession codes autoreactivity. Colour development was assayed after 19 h. Protein Data Bank: Coordinates and structure factors for GGA1- VHS:peptide complexes have been deposited with accession numbers of 3G2S (VHS:Sa complex), 3G2T (VHS:SaP complex), 3G2U (VHS:So Gel filtration complex), 3G2V (VHS:SoP complex) and 3G2W (VHS:HiP complex). A Superose 6 10/300 column (1.8–55 cm) was connected to a Fast Protein Liquid Chromatography (FPLC) machine (Amersham Biosciences). The column was equilibrated in 100 mM NaCl, 50 mM Tris–HCl pH 7.5 and Acknowledgments Sigma gel filtration molecular weight standards were used to calibrate the column before injecting the samples at varying concentrations (from [c] to We are grateful to L. Sottrup-Jensen and L. Kristensen for help to determine [c]/20; [c] = 7.0 mg/mL). Two-hundred and fifty microlitres of the protein protein concentration by total amino acid analysis and C. Olesen and M. sample (GGA1 wt, GGA1-short, S355A and S355D) was injected at room Blaise for collecting data on the VHS:SaP complex. Help from staff at the temperature with a flow rate of 0.25 mL/min. For complex samples, the synchrotrons at ESRF, European Molecular Biology Laboratory outstation GGA1 proteins were mixed with the Sortilin tail at a molar ratio of 1:4 and at Deutsches Elektronen Synchrotron (EMBL/DESY) and the Swiss Light ◦ incubated at 4 C before gel filtration. Fractions of 1 mL were collected and Source (SLS) is also gratefully acknowledged. For financial support, we an aliquot of each was subjected reducing SDS–PAGE on a 12% gel and would like to thank the Lundbeck Foundation and Danscatt. stained with Coomassie Brilliant Blue followed by destaining with 30% methanol, 10% acetic acid. Supporting Information Small angle X-ray scattering Additional Supporting Information may be found in the online version of SAXS analysis was carried out on GGA1 (wt), S355A and S355D in 100 mM this article: NaCl, 50 mM Tris–HCl (pH 7.5) primarily at a high [c]/2 and a low [c]/4 concentration. SAXS measurements were conducted with a laboratory- Figure S1: Comparison of peptides in the VHS binding pocket. A) based instrument at the University of Aarhus (45). Data were reduced The phosphorylated and nonphosphorylated peptides: So, SoP, Sa, SaP with the SUPERSAXS program package (C. L. P. O. and J. S. P., unpublished) and HiP are superimposed and shown as ball-and-stick model coloured and normalized to absolute scale using water as a primary standard. The by atom type (nitrogen, blue; carbon, grey; oxygen, red). The position Guinier plots [Ln(I)versusq2] shown in Supporting Information (insets of the conserved key residues are labelled and selected residues from the VHS binding pocket are shown (ball-and-stick model; carbon, yellow). in Figure S4A,C) show a linear behaviour at low q, which indicates B) Comparison of VHS:peptide interfaces between C-terminal tail of the monodispersity of the samples; from the slopes of the curves, one indicated cargo and the VHS domain from GGA1 and -3 calculated by the can obtain an estimation of the radius of gyration of particles in each PISA server http://www.ebi.ac.uk/msd-srv/prot int/pistart.html. sample. As a first step on the data analysis, we applied the indirect Fourier transformation (IFT) method (46). With this method it is possible to Figure S2: C-terminus recognition at the DXXLL site. The C-terminal calculate the pair distance distribution p(r) function, which is a histogram part of the DXXLL binding pocket shown in top view for GGA1-VHS of interpoint distances within the particles weighted by electron densities complexed with: (A) BACE-P(2) (purple), (B) So (yellow), (C) Sa (teal) and at the points (Figure S4A–E). From this analysis, it is possible to retrieve (D) CI-MPR(3) (green). The C-terminus ‘clamp’ at the end of the binding a more accurate estimation for the particle radius of gyration. The values pocket (consisting of Q142, S99, P100, K101 and Y102) is shown by obtained in this way are similar to the ones obtained from the Guinier surface representation coloured by atom type (nitrogen, blue; oxygen, red; analysis. Interestingly, the p(r) functions revealed similar dimensions and carbon, grey). The width of the ‘clamp’, measured as minimum distance indicated dimerization at high concentration of all three proteins. As atomic between the Gln142 and Lys102 side chain, is shown and indicated with resolution structures of three domains in GGA1 were known, rigid body a dotted line. reconstructions were performed using the BUNCH program (47), with the GAE domain constrained in its dimeric form (31). The reported GAE-hinge Figure S3: Intrinsic tryptophan fluorescence of GGA1. A) Fluorescence interaction was not implemented to keep maximum flexibility of the spectra of GGA1 wt and mutants were obtained at the varying hinge region (31). Tests assuming monomeric conformation of the protein concentrations: c, c/2, c/4 and c/20, using an excitation wavelength of give poor fits and very open conformation of the protein domains (data 295 nm and emission measured between 315 and 375 nm. The proteins not shown). The known atomic resolution models are connected with are shown as wt (solid), S355A (filled circles), S355D (filled triangles) flexible linkers and the structure is optimized using a simulated annealing and GGA1-short (dotted). B) Table with wavelength of the maximum procedure. As a result, the three-dimensional arrangement of the domains fluorescence and the relative emission of the GGA1 proteins. that gives the best fit of the experimental data is obtained. Ten independent simulations were performed for each of the studied GGA1 proteins. In Figure S4: SAXS data. SAXS was carried out at GGA1 wt (black), GGA1- each case, the calculated models were very similar, indicating a stable S355D (blue) and GGA1-S355A (red) at the concentrations c/2 and c/4.

Trafﬁc 2010; 11: 259–273 271 Cramer et al.

A) IFT fitting of wt, S355D and S355A all at the concentration c/2. Insert: 11. Takatsu H, Yoshino K, Toda K, Nakayama K. GGA proteins associate Guinier plot of wt, S355D and S355A. B) Pair distances distribution function with Golgi membranes through interaction between their GGAH at the concentration c/2. C) IFT fitting of wt, S355D and S355A mutation domains and ADP-ribosylation factors. Biochem J 2002;365:369–378. at a concentration of c/4. Insert: Gunier plot of wt, S355D and S355A. D) 12. Nielsen MS, Madsen P, Christensen EI, Nykjaer A, Gliemann J, Pair distances distribution function at the concentration c/4. E) Kratky plot Kasper D, Pohlmann R, Petersen CM. The sortilin cytoplasmic tail with information on compactness of wt, S355A and S355D. The curves conveys Golgi-endosome transport and binds the VHS domain of the for the GGA proteins indicate that the proteins are compact but contain GGA2 sorting protein. EMBO J 2001;20:2180–2190. flexible parts, as it does not show the complete bell-shaped structure 13. Puertollano R, Aguilar RC, Gorshkova I, Crouch RJ, Bonifacino JS. characterizing a completely compact molecule. F) Fits using rigid body Sorting of mannose 6-phosphate receptors mediated by the GGAs. modelling with the program BUNCH, assuming a dimeric conformation at Science 2001;292:1712–1716. the concentration c/2. 14. Takatsu H, Katoh Y, Shiba Y, Nakayama K. Golgi-localizing, gamma- adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) Figure S5: Solution structure of GGA for another conformation of the proteins interact with acidic dileucine sequences within the cytoplas- dimerization centre. A) Rigid body modelling of the GGA1 dimer (wt) at mic domains of sorting receptors through their Vps27p/Hrs/STAM concentration c/2. B) Model of GGA1 dimer in solution. The average of (VHS) domains. J Biol Chem 2001;276:28541–28545. the 10 runs is shown as semitransparent beads and the best model is 15. Zhu Y, Doray B, Poussu A, Lehto VP, Kornfeld S. Binding of GGA2 superimposed on the average structure. The three known domains are to the lysosomal enzyme sorting motif of the mannose 6-phosphate shown; the VHS domain (yellow, PDB ID: 1JWF), the GAT domain (blue, receptor. Science 2001;292:1716–1718. PDB ID: 1O3X) and the GAE (pale green/green, PDB ID: 1NA8). The model 16. Doray B, Ghosh P, Griffith J, Geuze HJ, Kornfeld S. Cooperation ◦ is rotated 90 .C)χ2 values for three different modelling approaches: of GGAs and AP-1 in packaging MPRs at the trans-Golgi network. dimerization centre given by structure 2DWY, dimerization centre given by Science (New York, NY 2002;297:1700–1703. structure 1NA8 and the model assuming monomeric conformation. The 17. Jacobsen L, Madsen P, Nielsen MS, Geraerts WP, Gliemann J, Smit resulting structures for the monomeric models are very open and therefore AB, Petersen CM. The sorLA cytoplasmic domain interacts with GGA1 physically unlikely. D) Theoretical scattering curves for the two possible and −2 and defines minimum requirements for GGA binding. FEBS conformations of the dimerization centre. letters 2002;511:155–158. 18. Nielsen MS, Gustafsen C, Madsen P, Nyengaard JR, Hermey G, Bakke Table S1: Data collection and processing O, Mari M, Schu P, Pohlmann R, Dennes A, Petersen CM. Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA. Molecular and cellular biology 2007;27:6842–6851. Table S2: Refinement statistics 19. Doray B, Bruns K, Ghosh P, Kornfeld S. Interaction of the cation- Please note: Wiley-Blackwell are not responsible for the content or dependent mannose 6-phosphate receptor with GGA proteins. 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