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SNX4 in complex with clathrin and dynein: implications for endosome movement.

Skånland SS, Wälchli S, Brech A, Sandvig K - PLoS ONE (2009)

Bottom Line: The interactions were inhibited by wortmannin, a PI3-kinase inhibitor, suggesting that they form when SNX4 is associated with PI(3)P on endosomes.Moreover, clathrin knockdown led to increased Golgi transport of the toxin ricin, as well as redistribution of endosomes.Upon clathrin release, dynein may bind SNX4 and mediate retrograde movement.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biomedicine, Faculty Division Norwegian Radium Hospital, University of Oslo, Oslo, Norway.

ABSTRACT

Background: Sorting nexins (SNXs) constitute a family of proteins classified by their phosphatidylinositol (PI) binding Phox homology (PX) domain. Some members regulate intracellular trafficking. We have here investigated mechanisms underlying SNX4 mediated endosome to Golgi transport.

Methodology/principal findings: We show that SNX4 forms complexes with clathrin and dynein. The interactions were inhibited by wortmannin, a PI3-kinase inhibitor, suggesting that they form when SNX4 is associated with PI(3)P on endosomes. We further localized the clathrin interacting site on SNX4 to a clathrin box variant. A short peptide containing this motif was sufficient to pull down both clathrin and dynein. Knockdown studies demonstrated that clathrin is not required for the SNX4/dynein interaction. Moreover, clathrin knockdown led to increased Golgi transport of the toxin ricin, as well as redistribution of endosomes.

Conclusions/significance: We discuss the possibility of clathrin serving as a regulator of SNX4-dependent transport. Upon clathrin release, dynein may bind SNX4 and mediate retrograde movement.

Show MeSH
SNX4 interacts with CHC via a clathrin box variant.(a) Schematic view of SNX4 wt, SNX4 198-451Δ and SNX4 109-113Δ. (b) Cells transfected as indicated were lysed and proceeded to anti-myc immunoprecipitation for 4 h at 4°C. The immunoprecipitate and whole cell lysate (WCL) were separated by SDS-PAGE and analyzed by Western blot with the indicated antibodies. Quantification of CHC binding corrected for expression level of the myc constructs is shown to the right, n = 3. (c) The amino acid sequences of GST-peptides are shown with putative clathrin box in red. The numbering represents amino acid number in the SNX4 sequence. GST pull-down was performed with cell lysate at 4°C overnight. The pull-down was subjected to SDS-PAGE and analyzed by Western blot with the indicated antibodies. GST alone was used as negative control. (d) Putative clathrin boxes of SNX1, SNX2, SNX3, SNX4 (105–116) and reverse clathrin box of Hrs are aligned. GST pull-down was performed as in (c) with the indicated peptides. (e) Schematic view of GST-peptides containing the putative clathrin box (red) and point mutations (blue). GST pull-down was performed as in (c) with the indicated peptides.
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pone-0005935-g003: SNX4 interacts with CHC via a clathrin box variant.(a) Schematic view of SNX4 wt, SNX4 198-451Δ and SNX4 109-113Δ. (b) Cells transfected as indicated were lysed and proceeded to anti-myc immunoprecipitation for 4 h at 4°C. The immunoprecipitate and whole cell lysate (WCL) were separated by SDS-PAGE and analyzed by Western blot with the indicated antibodies. Quantification of CHC binding corrected for expression level of the myc constructs is shown to the right, n = 3. (c) The amino acid sequences of GST-peptides are shown with putative clathrin box in red. The numbering represents amino acid number in the SNX4 sequence. GST pull-down was performed with cell lysate at 4°C overnight. The pull-down was subjected to SDS-PAGE and analyzed by Western blot with the indicated antibodies. GST alone was used as negative control. (d) Putative clathrin boxes of SNX1, SNX2, SNX3, SNX4 (105–116) and reverse clathrin box of Hrs are aligned. GST pull-down was performed as in (c) with the indicated peptides. (e) Schematic view of GST-peptides containing the putative clathrin box (red) and point mutations (blue). GST pull-down was performed as in (c) with the indicated peptides.

Mentions: A recognized CHC interacting motif is the so-called clathrin box LΦpΦ(−), where L is leucine, Φ a bulky hydrophobic residue, p a polar residue, and (−) is a negatively charged residue [23]. Amino acid sequence analysis of SNX4 did not show such a motif. However, a variant, or an “inverted” clathrin box, 109EFELL, was found in its PX domain. We therefore chose to test whether this sequence was required for CHC binding. We constructed the deletion mutant SNX4 109-113Δ and also designed a SNX4 construct deleted of its C-terminal half, SNX4 198-451Δ (Figure 3a). Both wild-type SNX4 and the two mutants were designed with a myc-tag. Since a mutation in the PX domain could potentially affect the endosomal localization of SNX4, we first investigated the cellular distribution of the recombinant SNX4 proteins by immunofluorescence. No apparent change in localization was observed for SNX4 109-113Δ compared to SNX4 wt (Figure S2a), but its co-localization with EEA1 was reduced to 67% relative to the wild type (n = 6 for SNX4 wt and 8 for SNX4 109-113Δ) (Figure S2b). SNX4 198-451Δ was for an unexplained reason present in the nucleus in addition to its expected cytoplasmic localization (Figure S2a). We next performed anti-myc immunoprecipitation experiments on cells expressing the SNX4 constructs, and examined the presence of CHC. As shown in Figure 3b, a mild reduction in the CHC binding was observed for SNX4 198-451Δ, whereas it was practically abolished for the SNX4 109-113Δ mutant. Three independent experiments were quantified and corrected for SNX4 expression level. The result showed an ∼80% reduction in CHC binding for SNX4 109-113Δ compared to SNX4 wt (Figure 3b). This suggests that the clathrin box variant present in SNX4 may be a CHC binding site. In order to validate this, we designed GST-peptides from the SNX4 sequence containing the putative CHC binding motif. We made one peptide containing amino acids 101–132, and a shorter one from amino acid 105–116 (Figure 3c). The peptides were purified and added to cell lysate before pull-down. CHC was present in both the 101–132 and 105–116 pull-downs (Figure 3c). GST alone was used as negative control and was unable to pull down CHC (Figure 3c). Sequences corresponding to 109EFELL in SNX4 are also present in other SNXs. We therefore investigated whether these sequences from SNX1, SNX2 and SNX3 could also pull down CHC. A sequence containing the clathrin box of Hrs was used as positive control [20]. The clathrin box of Hrs is represented from the C- to the N-terminal end (rev) and aligned with the putative clathrin boxes of the SNXs in Figure 3d. The main residues are conserved. In pull-down experiments all the peptides, but not GST alone, were able to bind CHC (Figure 3d). In order to study the contribution of the different amino acids of 109EFELL, we performed an “alanine scan” of the short peptide. We exchanged the five amino acids one by one with alanine (Figure 3e), and performed pull-down experiments with these peptides. We observed that the E109A mutant was still able to pull down CHC, perhaps more efficiently than the wild type, while the mutants F110A, E111A, L112A and L113A only showed background binding to CHC (Figure 3e). Interestingly, F110 and L113 are strictly conserved in all the putative clathrin boxes tested (Figure 3d), and might represent necessary residues. From these data we conclude that the SNX4 clathrin box variant is important for CHC binding.


SNX4 in complex with clathrin and dynein: implications for endosome movement.

Skånland SS, Wälchli S, Brech A, Sandvig K - PLoS ONE (2009)

SNX4 interacts with CHC via a clathrin box variant.(a) Schematic view of SNX4 wt, SNX4 198-451Δ and SNX4 109-113Δ. (b) Cells transfected as indicated were lysed and proceeded to anti-myc immunoprecipitation for 4 h at 4°C. The immunoprecipitate and whole cell lysate (WCL) were separated by SDS-PAGE and analyzed by Western blot with the indicated antibodies. Quantification of CHC binding corrected for expression level of the myc constructs is shown to the right, n = 3. (c) The amino acid sequences of GST-peptides are shown with putative clathrin box in red. The numbering represents amino acid number in the SNX4 sequence. GST pull-down was performed with cell lysate at 4°C overnight. The pull-down was subjected to SDS-PAGE and analyzed by Western blot with the indicated antibodies. GST alone was used as negative control. (d) Putative clathrin boxes of SNX1, SNX2, SNX3, SNX4 (105–116) and reverse clathrin box of Hrs are aligned. GST pull-down was performed as in (c) with the indicated peptides. (e) Schematic view of GST-peptides containing the putative clathrin box (red) and point mutations (blue). GST pull-down was performed as in (c) with the indicated peptides.
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pone-0005935-g003: SNX4 interacts with CHC via a clathrin box variant.(a) Schematic view of SNX4 wt, SNX4 198-451Δ and SNX4 109-113Δ. (b) Cells transfected as indicated were lysed and proceeded to anti-myc immunoprecipitation for 4 h at 4°C. The immunoprecipitate and whole cell lysate (WCL) were separated by SDS-PAGE and analyzed by Western blot with the indicated antibodies. Quantification of CHC binding corrected for expression level of the myc constructs is shown to the right, n = 3. (c) The amino acid sequences of GST-peptides are shown with putative clathrin box in red. The numbering represents amino acid number in the SNX4 sequence. GST pull-down was performed with cell lysate at 4°C overnight. The pull-down was subjected to SDS-PAGE and analyzed by Western blot with the indicated antibodies. GST alone was used as negative control. (d) Putative clathrin boxes of SNX1, SNX2, SNX3, SNX4 (105–116) and reverse clathrin box of Hrs are aligned. GST pull-down was performed as in (c) with the indicated peptides. (e) Schematic view of GST-peptides containing the putative clathrin box (red) and point mutations (blue). GST pull-down was performed as in (c) with the indicated peptides.
Mentions: A recognized CHC interacting motif is the so-called clathrin box LΦpΦ(−), where L is leucine, Φ a bulky hydrophobic residue, p a polar residue, and (−) is a negatively charged residue [23]. Amino acid sequence analysis of SNX4 did not show such a motif. However, a variant, or an “inverted” clathrin box, 109EFELL, was found in its PX domain. We therefore chose to test whether this sequence was required for CHC binding. We constructed the deletion mutant SNX4 109-113Δ and also designed a SNX4 construct deleted of its C-terminal half, SNX4 198-451Δ (Figure 3a). Both wild-type SNX4 and the two mutants were designed with a myc-tag. Since a mutation in the PX domain could potentially affect the endosomal localization of SNX4, we first investigated the cellular distribution of the recombinant SNX4 proteins by immunofluorescence. No apparent change in localization was observed for SNX4 109-113Δ compared to SNX4 wt (Figure S2a), but its co-localization with EEA1 was reduced to 67% relative to the wild type (n = 6 for SNX4 wt and 8 for SNX4 109-113Δ) (Figure S2b). SNX4 198-451Δ was for an unexplained reason present in the nucleus in addition to its expected cytoplasmic localization (Figure S2a). We next performed anti-myc immunoprecipitation experiments on cells expressing the SNX4 constructs, and examined the presence of CHC. As shown in Figure 3b, a mild reduction in the CHC binding was observed for SNX4 198-451Δ, whereas it was practically abolished for the SNX4 109-113Δ mutant. Three independent experiments were quantified and corrected for SNX4 expression level. The result showed an ∼80% reduction in CHC binding for SNX4 109-113Δ compared to SNX4 wt (Figure 3b). This suggests that the clathrin box variant present in SNX4 may be a CHC binding site. In order to validate this, we designed GST-peptides from the SNX4 sequence containing the putative CHC binding motif. We made one peptide containing amino acids 101–132, and a shorter one from amino acid 105–116 (Figure 3c). The peptides were purified and added to cell lysate before pull-down. CHC was present in both the 101–132 and 105–116 pull-downs (Figure 3c). GST alone was used as negative control and was unable to pull down CHC (Figure 3c). Sequences corresponding to 109EFELL in SNX4 are also present in other SNXs. We therefore investigated whether these sequences from SNX1, SNX2 and SNX3 could also pull down CHC. A sequence containing the clathrin box of Hrs was used as positive control [20]. The clathrin box of Hrs is represented from the C- to the N-terminal end (rev) and aligned with the putative clathrin boxes of the SNXs in Figure 3d. The main residues are conserved. In pull-down experiments all the peptides, but not GST alone, were able to bind CHC (Figure 3d). In order to study the contribution of the different amino acids of 109EFELL, we performed an “alanine scan” of the short peptide. We exchanged the five amino acids one by one with alanine (Figure 3e), and performed pull-down experiments with these peptides. We observed that the E109A mutant was still able to pull down CHC, perhaps more efficiently than the wild type, while the mutants F110A, E111A, L112A and L113A only showed background binding to CHC (Figure 3e). Interestingly, F110 and L113 are strictly conserved in all the putative clathrin boxes tested (Figure 3d), and might represent necessary residues. From these data we conclude that the SNX4 clathrin box variant is important for CHC binding.

Bottom Line: The interactions were inhibited by wortmannin, a PI3-kinase inhibitor, suggesting that they form when SNX4 is associated with PI(3)P on endosomes.Moreover, clathrin knockdown led to increased Golgi transport of the toxin ricin, as well as redistribution of endosomes.Upon clathrin release, dynein may bind SNX4 and mediate retrograde movement.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biomedicine, Faculty Division Norwegian Radium Hospital, University of Oslo, Oslo, Norway.

ABSTRACT

Background: Sorting nexins (SNXs) constitute a family of proteins classified by their phosphatidylinositol (PI) binding Phox homology (PX) domain. Some members regulate intracellular trafficking. We have here investigated mechanisms underlying SNX4 mediated endosome to Golgi transport.

Methodology/principal findings: We show that SNX4 forms complexes with clathrin and dynein. The interactions were inhibited by wortmannin, a PI3-kinase inhibitor, suggesting that they form when SNX4 is associated with PI(3)P on endosomes. We further localized the clathrin interacting site on SNX4 to a clathrin box variant. A short peptide containing this motif was sufficient to pull down both clathrin and dynein. Knockdown studies demonstrated that clathrin is not required for the SNX4/dynein interaction. Moreover, clathrin knockdown led to increased Golgi transport of the toxin ricin, as well as redistribution of endosomes.

Conclusions/significance: We discuss the possibility of clathrin serving as a regulator of SNX4-dependent transport. Upon clathrin release, dynein may bind SNX4 and mediate retrograde movement.

Show MeSH