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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 inhibits EGFR transport and degradation without affecting retrograde and recycling transport routes.(A-C) After cell surface binding, EGF-biotin coupled to streptavidinAlexaFluor488 was endocytosed for 10 min (A) or 50 min (B-C) at 37°C in HeLa cells expressing mRFP1-SNX12. Cells were labeled with anti-EEA1 (A) or Lamp1 (B-C) antibodies and analyzed by triple channel fluorescence. (D) HeLa cells expressing myc-SNX12 or myc-SNX3 or mock-treated were incubated with EGF for the indicated time periods. Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and western blotting with antibodies against EGFR, α-tubulin (a-tub) or myc. (E) After cell surface binding, Shiga toxin B-subunit conjugated to Cy3 was internalized for 10 min or 50 min at 37°C into HeLa cells expressing GFP-SNX12. Cells were labeled with anti-transferrin receptor (TfR) or Rab6 antibodies and analyzed by triple channel fluorescence. (F) After cell surface binding (0 min), transferrin conjugated to AlexaFluor546 (transferrin546) was internalized for 30 min or 120 min at 37°C into control cells (upper panels) or cells expressing GFP-SNX12 (lower panels). Cells were then analyzed by fluorescence. (A-C and E-F) Scale bar indicates 10 µm.
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pone-0038949-g002: SNX12 overexpression inhibits EGFR transport and degradation without affecting retrograde and recycling transport routes.(A-C) After cell surface binding, EGF-biotin coupled to streptavidinAlexaFluor488 was endocytosed for 10 min (A) or 50 min (B-C) at 37°C in HeLa cells expressing mRFP1-SNX12. Cells were labeled with anti-EEA1 (A) or Lamp1 (B-C) antibodies and analyzed by triple channel fluorescence. (D) HeLa cells expressing myc-SNX12 or myc-SNX3 or mock-treated were incubated with EGF for the indicated time periods. Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and western blotting with antibodies against EGFR, α-tubulin (a-tub) or myc. (E) After cell surface binding, Shiga toxin B-subunit conjugated to Cy3 was internalized for 10 min or 50 min at 37°C into HeLa cells expressing GFP-SNX12. Cells were labeled with anti-transferrin receptor (TfR) or Rab6 antibodies and analyzed by triple channel fluorescence. (F) After cell surface binding (0 min), transferrin conjugated to AlexaFluor546 (transferrin546) was internalized for 30 min or 120 min at 37°C into control cells (upper panels) or cells expressing GFP-SNX12 (lower panels). Cells were then analyzed by fluorescence. (A-C and E-F) Scale bar indicates 10 µm.

Mentions: Previously we had observed that excess SNX3 inhibited membrane transport beyond early endosomes. We then tested whether SNX12 overexpression caused a similar inhibition. When overexpressed, SNX12 did not affect EGF internalization since endocytosed EGFAlexaFluor488 colocalized with EEA1-positive early endosomes after 10 min pulse (Figure 2A) much like in control cells (Figure S1A). However, and in contrast to control cells (Figure S1B-C), EGF was not transported to Lamp1-positive late endosomes (Figure 2C) but still remained in early endosomes (Figure 2B) after 50 min in cells expressing SNX12. Consistent with these observations, overexpressed Myc-SNX12 inhibited EGFR degradation in the presence of EGF to the same extent as myc-SNX3, at comparable levels of expression (Figure 2D). By contrast, the PtdIns3P binding-defective mutant SNX12R71A did not affect EGFR degradation (Figure S2D). This demonstrates that SNX12R71A mutant, which is not able to bind PtdIns3P, having lost the capacity to localize to endosomes, cannot interfere EGFR transport and degradation.


SNX12 role in endosome membrane transport.

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

SNX12 overexpression inhibits EGFR transport and degradation without affecting retrograde and recycling transport routes.(A-C) After cell surface binding, EGF-biotin coupled to streptavidinAlexaFluor488 was endocytosed for 10 min (A) or 50 min (B-C) at 37°C in HeLa cells expressing mRFP1-SNX12. Cells were labeled with anti-EEA1 (A) or Lamp1 (B-C) antibodies and analyzed by triple channel fluorescence. (D) HeLa cells expressing myc-SNX12 or myc-SNX3 or mock-treated were incubated with EGF for the indicated time periods. Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and western blotting with antibodies against EGFR, α-tubulin (a-tub) or myc. (E) After cell surface binding, Shiga toxin B-subunit conjugated to Cy3 was internalized for 10 min or 50 min at 37°C into HeLa cells expressing GFP-SNX12. Cells were labeled with anti-transferrin receptor (TfR) or Rab6 antibodies and analyzed by triple channel fluorescence. (F) After cell surface binding (0 min), transferrin conjugated to AlexaFluor546 (transferrin546) was internalized for 30 min or 120 min at 37°C into control cells (upper panels) or cells expressing GFP-SNX12 (lower panels). Cells were then analyzed by fluorescence. (A-C and E-F) Scale bar indicates 10 µm.
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Related In: Results  -  Collection

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pone-0038949-g002: SNX12 overexpression inhibits EGFR transport and degradation without affecting retrograde and recycling transport routes.(A-C) After cell surface binding, EGF-biotin coupled to streptavidinAlexaFluor488 was endocytosed for 10 min (A) or 50 min (B-C) at 37°C in HeLa cells expressing mRFP1-SNX12. Cells were labeled with anti-EEA1 (A) or Lamp1 (B-C) antibodies and analyzed by triple channel fluorescence. (D) HeLa cells expressing myc-SNX12 or myc-SNX3 or mock-treated were incubated with EGF for the indicated time periods. Cell lysates (100 µg) were analyzed by SDS gel electrophoresis and western blotting with antibodies against EGFR, α-tubulin (a-tub) or myc. (E) After cell surface binding, Shiga toxin B-subunit conjugated to Cy3 was internalized for 10 min or 50 min at 37°C into HeLa cells expressing GFP-SNX12. Cells were labeled with anti-transferrin receptor (TfR) or Rab6 antibodies and analyzed by triple channel fluorescence. (F) After cell surface binding (0 min), transferrin conjugated to AlexaFluor546 (transferrin546) was internalized for 30 min or 120 min at 37°C into control cells (upper panels) or cells expressing GFP-SNX12 (lower panels). Cells were then analyzed by fluorescence. (A-C and E-F) Scale bar indicates 10 µm.
Mentions: Previously we had observed that excess SNX3 inhibited membrane transport beyond early endosomes. We then tested whether SNX12 overexpression caused a similar inhibition. When overexpressed, SNX12 did not affect EGF internalization since endocytosed EGFAlexaFluor488 colocalized with EEA1-positive early endosomes after 10 min pulse (Figure 2A) much like in control cells (Figure S1A). However, and in contrast to control cells (Figure S1B-C), EGF was not transported to Lamp1-positive late endosomes (Figure 2C) but still remained in early endosomes (Figure 2B) after 50 min in cells expressing SNX12. Consistent with these observations, overexpressed Myc-SNX12 inhibited EGFR degradation in the presence of EGF to the same extent as myc-SNX3, at comparable levels of expression (Figure 2D). By contrast, the PtdIns3P binding-defective mutant SNX12R71A did not affect EGFR degradation (Figure S2D). This demonstrates that SNX12R71A mutant, which is not able to bind PtdIns3P, having lost the capacity to localize to endosomes, cannot interfere EGFR transport and degradation.

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