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. 2009 Jun 16;4(6):e5935.
doi: 10.1371/journal.pone.0005935.

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

Sigrid S Skånland  1 , Sébastien Wälchli, Andreas Brech, Kirsten Sandvig
Affiliations

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

Sigrid S Skånland et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SNX4 co-immunoprecipitates CHC, tubulin and dynein.
(a) HEK293 cells were treated with or without 100 nM wortmannin (Wort.) for 30 min, or transfected with control or SNX4 siRNA for 72 h, before lysis and anti-SNX4 immunoprecipitation at 4°C overnight. As a negative control, cell lysate was treated without antibody. The immunoprecipitate and whole cell lysate (WCL) were then separated by SDS-PAGE and analyzed by Western blot with the indicated antibodies. (b) Cells were transfected with control or SNX4 siRNA for 72 h before fixation and staining with anti-CHC antibodies. (c) Cells were treated with or without 100 nM wortmannin for 30 min before fixation and staining with anti-tubulin antibodies. (d) Cells were treated with or without 20 µM nocodazole for 30 min before fixation and antibody staining. DRAQ5 was used to stain the nuclei. Bars, 10 µm (b) and 5 µm (c, d).
Figure 2
Figure 2. GFP-SNX4 co-localizes with tubulin and CHC.
(a–b) Cells were transiently transfected with GFP-SNX4 for 24 h before the cells were fixed and stained as indicated. (c) Cells expressing high levels of GFP-SNX4 were fixed and stained as indicated. DRAQ5 was used to stain the nuclei. Bars, 5 µm.
Figure 3
Figure 3. 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.
Figure 4
Figure 4. The SNX4 interaction with dynein does not require CHC.
(a) Cells were transfected with control siRNA or siRNA targeting CHC (siCHC) as described in Materials and Methods . Lysate of the cells was then subjected to SNX4 immunoprecipitation at 4°C overnight, and the precipitate separated by SDS-PAGE before Western blot analysis with the indicated antibodies. (b) Cells were treated with or without nocodazole for 30 min before lysis and SNX4 immunoprecipitation. The precipitate was separated by SDS-PAGE and analysed by Western blot with the indicated antibodies. (c) GST pull-down of 101–132, 105–116 or GST alone 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. (d) Cells transfected as indicated were lysed and proceeded to anti-myc immunoprecipitation for 3 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.
Figure 5
Figure 5. CHC knockdown leads to increased ricin transport.
(a) Transfected cells were lysed and analyzed by Western blot with the indicated antibodies. (b) Cells transfected as indicated were incubated with radioactive sulphate for 3 h before further incubation with ricin sulf-1 for 90 min and subsequent lysis. Ricin was immunoprecipitated from the lysates, and the precipitate separated by SDS-PAGE before autoradiography. The intensity of the bands was quantified and the average plotted with error bars showing standard deviations. (c) Transfected cells were incubated with [3H]mannose in glucose free medium for 3 h before further incubation with ricin sulf-2 for 3 h. Cell lysate was immunoprecipitated with anti-ricin antibodies. The precipitate was analyzed by autoradiography. (d) Cells transfected as indicated were incubated with 2 µg/ml ricin for 45 min before fixation and staining with antibodies as indicated. Bars, 5 µm. (e) Cells transfected with siCHCv were incubated with 2 µg/ml ricin for 45 min before fixation and staining with the indicated antibodies. Asterisks in the merged picture indicate control cells. Bar, 5 µm.
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