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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
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.
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pone-0005935-g002: 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.

Mentions: Since the functional antibodies for use in immunofluorescence experiments were all raised in mice, we could not perform co-localization studies with endogenous SNX4 and its binding partners. To circumvent this, we cloned and expressed SNX4 as a GFP fusion protein. In agreement with the co-immunoprecipitation experiments, we observed GFP-SNX4 positive vesicles in close proximity to, or overlapping with, tubulin (Figure 2a). In the case of CHC, we observed a clear co-localization with GFP-SNX4, and even triple co-localization with the early endosomal protein EEA1 (Figure 2b). The fraction of GFP-SNX4 that co-localized with CHC at moderate expression level was quantified to be 27.8±6.6% (average with standard deviation, n = 15), and with tubulin 19.4±7.6% (average with standard deviation, n = 5). In order to exclude the possibility that this co-localization was random, we expressed GFP alone and performed the corresponding experiments. Even though GFP is expressed throughout the cell, less than 0.5% co-localization could be detected with CHC (n = 16) or tubulin (n = 10) (pictures not presented). This clearly demonstrates that the co-localization between GFP-SNX4 and CHC or tubulin is significant (p<0.0001 by Student's t-test). At high GFP-SNX4 expression level, CHC appeared more recruited to GFP-SNX4 positive vesicles (Figure 2c). We furthermore confirmed that endogenous SNX4 partly co-localized with transiently expressed GFP-CLC (clathrin light chain) (13.2±1.9%; average with standard deviation, n = 10) (Figure S1). These data support the suggestion of SNX4/tubulin and SNX4/CHC complexes on endosomes.


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

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

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.
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Related In: Results  -  Collection

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pone-0005935-g002: 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.
Mentions: Since the functional antibodies for use in immunofluorescence experiments were all raised in mice, we could not perform co-localization studies with endogenous SNX4 and its binding partners. To circumvent this, we cloned and expressed SNX4 as a GFP fusion protein. In agreement with the co-immunoprecipitation experiments, we observed GFP-SNX4 positive vesicles in close proximity to, or overlapping with, tubulin (Figure 2a). In the case of CHC, we observed a clear co-localization with GFP-SNX4, and even triple co-localization with the early endosomal protein EEA1 (Figure 2b). The fraction of GFP-SNX4 that co-localized with CHC at moderate expression level was quantified to be 27.8±6.6% (average with standard deviation, n = 15), and with tubulin 19.4±7.6% (average with standard deviation, n = 5). In order to exclude the possibility that this co-localization was random, we expressed GFP alone and performed the corresponding experiments. Even though GFP is expressed throughout the cell, less than 0.5% co-localization could be detected with CHC (n = 16) or tubulin (n = 10) (pictures not presented). This clearly demonstrates that the co-localization between GFP-SNX4 and CHC or tubulin is significant (p<0.0001 by Student's t-test). At high GFP-SNX4 expression level, CHC appeared more recruited to GFP-SNX4 positive vesicles (Figure 2c). We furthermore confirmed that endogenous SNX4 partly co-localized with transiently expressed GFP-CLC (clathrin light chain) (13.2±1.9%; average with standard deviation, n = 10) (Figure S1). These data support the suggestion of SNX4/tubulin and SNX4/CHC complexes on endosomes.

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