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SNX12 role in endosome membrane transport.

Pons V, Ustunel C, Rolland C, Torti E, Parton RG, Gruenberg J - PLoS ONE (2012)

Bottom Line: We found that SNX12 is expressed at very low levels compared to SNX3.SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides.Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Geneva, Switzerland.

ABSTRACT
In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

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SNX12 overexpression induces an accumulation of MVB-like structures and rescues the intralumenal vesicles that incorporate the EGF receptor.(A-B) HeLa cells expressing GFP-SNX12 were fixed and processed for electron microscopy. Cryosections were labeled with antibodies against GFP followed by protein A-gold 10 nm (arrows). Panel A shows a cluster of several multivesicular endosomes, each being labeled with a star, and panel B shows a high magnification view of an individual multivesicular endosome. Scale bar indicates 250 nm. (C) Hrs (D-E) or SNX3 (F-G) was knocked down and mRFP-SNX12 (red) was overexpressed (E-G) or not (D-F). In each condition, HeLa cells were also transfected with GFP-Rab5Q79L (green) during the last 24 h. After cell surface binding, EGF was internalized for 15 min at 37°C. Cells were processed for immunofluorescence with anti-EGFR antibodies (blue). The relative amount of EGFR in the lumen of endosome was quantified as previously described [25]. Results are expressed as the percentage of the total amount of EGFR. Each condition is the mean of at least three independent experiments; standard errors are indicated and results were analyzed by paired t test (***, p<0,001). (D-G) Representative pictures of experiments quantified in (C). GFP-Rab5Q79L enlarged endosomes in each condition showed the EGFR localization after Hrs (D-E) or SNX3 (F-G) was knocked down and mRFP-SNX12 (red) was overexpressed (E-G) or not (D-F).
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pone-0038949-g004: SNX12 overexpression induces an accumulation of MVB-like structures and rescues the intralumenal vesicles that incorporate the EGF receptor.(A-B) HeLa cells expressing GFP-SNX12 were fixed and processed for electron microscopy. Cryosections were labeled with antibodies against GFP followed by protein A-gold 10 nm (arrows). Panel A shows a cluster of several multivesicular endosomes, each being labeled with a star, and panel B shows a high magnification view of an individual multivesicular endosome. Scale bar indicates 250 nm. (C) Hrs (D-E) or SNX3 (F-G) was knocked down and mRFP-SNX12 (red) was overexpressed (E-G) or not (D-F). In each condition, HeLa cells were also transfected with GFP-Rab5Q79L (green) during the last 24 h. After cell surface binding, EGF was internalized for 15 min at 37°C. Cells were processed for immunofluorescence with anti-EGFR antibodies (blue). The relative amount of EGFR in the lumen of endosome was quantified as previously described [25]. Results are expressed as the percentage of the total amount of EGFR. Each condition is the mean of at least three independent experiments; standard errors are indicated and results were analyzed by paired t test (***, p<0,001). (D-G) Representative pictures of experiments quantified in (C). GFP-Rab5Q79L enlarged endosomes in each condition showed the EGFR localization after Hrs (D-E) or SNX3 (F-G) was knocked down and mRFP-SNX12 (red) was overexpressed (E-G) or not (D-F).

Mentions: Since SNX12 overexpression inhibited transport beyond early endosomes towards late endosomes and lysosomes, we analyzed whether the ultrastructure of the early endosomes was affected. By electron microscopy, clusters containing several multivesicular endosomes were typically observed in cells overexpressing SNX12 (Figure 4A), like SNX3 [25], while such clusters were not observed in control cells (not shown) [3]. Each individual multivesicular element, however, resembled ECV/MVBs, which function as transport intermediates between early and late endosomes. Immunogold labeling showed that the limiting membrane of these structures was decorated with GFP-SNX12, particularly in electron-dense flat regions of the limiting membrane (Figure 4B) resembling the endosomal clathrin coat involved in sorting ubiquitinated proteins into intralumenal vesicles of early endosomes [30], [31]. Since SNX12 is primarily located on early endosomes (Figure 1), these ECV-MVB-like structures presumably correspond to the vesicular regions of early endosomes that cannot detach or mature from early endosomes anymore, perhaps because SNX12 accumulation interferes with coat disassembly.


SNX12 role in endosome membrane transport.

Pons V, Ustunel C, Rolland C, Torti E, Parton RG, Gruenberg J - PLoS ONE (2012)

SNX12 overexpression induces an accumulation of MVB-like structures and rescues the intralumenal vesicles that incorporate the EGF receptor.(A-B) HeLa cells expressing GFP-SNX12 were fixed and processed for electron microscopy. Cryosections were labeled with antibodies against GFP followed by protein A-gold 10 nm (arrows). Panel A shows a cluster of several multivesicular endosomes, each being labeled with a star, and panel B shows a high magnification view of an individual multivesicular endosome. Scale bar indicates 250 nm. (C) Hrs (D-E) or SNX3 (F-G) was knocked down and mRFP-SNX12 (red) was overexpressed (E-G) or not (D-F). In each condition, HeLa cells were also transfected with GFP-Rab5Q79L (green) during the last 24 h. After cell surface binding, EGF was internalized for 15 min at 37°C. Cells were processed for immunofluorescence with anti-EGFR antibodies (blue). The relative amount of EGFR in the lumen of endosome was quantified as previously described [25]. Results are expressed as the percentage of the total amount of EGFR. Each condition is the mean of at least three independent experiments; standard errors are indicated and results were analyzed by paired t test (***, p<0,001). (D-G) Representative pictures of experiments quantified in (C). GFP-Rab5Q79L enlarged endosomes in each condition showed the EGFR localization after Hrs (D-E) or SNX3 (F-G) was knocked down and mRFP-SNX12 (red) was overexpressed (E-G) or not (D-F).
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Related In: Results  -  Collection

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pone-0038949-g004: SNX12 overexpression induces an accumulation of MVB-like structures and rescues the intralumenal vesicles that incorporate the EGF receptor.(A-B) HeLa cells expressing GFP-SNX12 were fixed and processed for electron microscopy. Cryosections were labeled with antibodies against GFP followed by protein A-gold 10 nm (arrows). Panel A shows a cluster of several multivesicular endosomes, each being labeled with a star, and panel B shows a high magnification view of an individual multivesicular endosome. Scale bar indicates 250 nm. (C) Hrs (D-E) or SNX3 (F-G) was knocked down and mRFP-SNX12 (red) was overexpressed (E-G) or not (D-F). In each condition, HeLa cells were also transfected with GFP-Rab5Q79L (green) during the last 24 h. After cell surface binding, EGF was internalized for 15 min at 37°C. Cells were processed for immunofluorescence with anti-EGFR antibodies (blue). The relative amount of EGFR in the lumen of endosome was quantified as previously described [25]. Results are expressed as the percentage of the total amount of EGFR. Each condition is the mean of at least three independent experiments; standard errors are indicated and results were analyzed by paired t test (***, p<0,001). (D-G) Representative pictures of experiments quantified in (C). GFP-Rab5Q79L enlarged endosomes in each condition showed the EGFR localization after Hrs (D-E) or SNX3 (F-G) was knocked down and mRFP-SNX12 (red) was overexpressed (E-G) or not (D-F).
Mentions: Since SNX12 overexpression inhibited transport beyond early endosomes towards late endosomes and lysosomes, we analyzed whether the ultrastructure of the early endosomes was affected. By electron microscopy, clusters containing several multivesicular endosomes were typically observed in cells overexpressing SNX12 (Figure 4A), like SNX3 [25], while such clusters were not observed in control cells (not shown) [3]. Each individual multivesicular element, however, resembled ECV/MVBs, which function as transport intermediates between early and late endosomes. Immunogold labeling showed that the limiting membrane of these structures was decorated with GFP-SNX12, particularly in electron-dense flat regions of the limiting membrane (Figure 4B) resembling the endosomal clathrin coat involved in sorting ubiquitinated proteins into intralumenal vesicles of early endosomes [30], [31]. Since SNX12 is primarily located on early endosomes (Figure 1), these ECV-MVB-like structures presumably correspond to the vesicular regions of early endosomes that cannot detach or mature from early endosomes anymore, perhaps because SNX12 accumulation interferes with coat disassembly.

Bottom Line: We found that SNX12 is expressed at very low levels compared to SNX3.SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides.Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Geneva, Switzerland.

ABSTRACT
In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

Show MeSH
Related in: MedlinePlus