SNX12 Role in Endosome Membrane Transport

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

Role of Sorting Nexins in intralumenal vesicle formation

In this study we investigated the function of Sorting Nexin 3 (SNX3) in the formation of intralumenal vesicles (ILVs). SNX3 is a small protein containing a particular PX domain that recognizes PI3P, hence the protein is located on the early endosomes, where it regulates ILV formation and retrograde transport of some cargoes. We investigated the role of SNX3 homologue SNX12 in the endocytic pathway and found that these proteins share redundant functions on ILV formation. In collaboration with Michael Overduin and Marc Lenoir we elucidated the solution structure of full-length SNX3 and identified a serine residue (Ser72) at the rim of the PI3P recognition pocket, which prevents PI3P binding once it is phosphorylated. Conserved nature of this serine residue among the SNX Family members suggests phosphorylation as a regulatory mechanism for PI3P-binding PX domains. To identify the signaling pathways involved in regulation of SNX3, we developed a kinase inhibitory library screen using HeLa cells stably expressing GFP-SNX3. Our screen revealed several potential targets, which regulate amount of GFP-SNX3 on endosomes

SNX12 is less expressed than SNX3 but is also localized on early endosomes.

<p>(<b>A</b>) Alignment of amino acid sequences of Homo sapiens SNX3 and SNX12. (<b>B</b>) RNA was extracted from different cell lines as indicated and relative amounts of endogenous SNX3 and SNX12 mRNA were quantified by RT-PCR. Values are indicated in the table below the graph since they are very low for SNX12 mRNA. (<b>C</b>) HeLa cells expressing GFP-SNX12 or co-expressing GFP-SNX12 and mRFP1-SNX3 were processed for immunofluorescence using the indicated antibodies. Scale bar indicates 10 µm.</p

SNX12 silencing has no effect on VSV infection and EGFR transport and degradation.

<p>(<b>A</b>) HeLa cells treated with siRNAs against SNX12 or mock-treated were lysed. Lysates were analyzed by SDS gel electrophoresis and western blotting using with antibodies against myc, SNX3 or actin. (<b>B-C</b>) HeLa cells were treated with SNX12 siRNAs or mock-treated, and then microtubules were depolymerized or not with 10 µM nocodazole for 2 h. VSV (1 MOI) was bound at 4°C at the cell surface and cells were then incubated for 3 h at 37°C to allow VSV infection to proceed. Cells were analyzed by immunofluorescence with antibodies against VSV-G protein. Scale bar indicates 10 µm. Experiments were quantified (<b>B</b>) and representative pictures were shown in (<b>C</b>). Under these conditions ≈50% of the control cells were infected so that changes in infection rate can be best monitored, and values are normalized to the controls. Each condition is the mean of at least three independent experiments; standard errors are indicated. (<b>D</b>) After cell surface binding, EGF-biotin coupled to streptavidin^{AlexaFluor488} was endocytosed for 10 min or 50 min at 37°C in HeLa cells treated siRNAs against SNX12. Cells were labeled with anti-EEA1 or Lamp1 antibodies and analyzed by triple channel fluorescence. (<b>E</b>) HeLa cells were treated with siRNAs against SNX12 or mock treated and then 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 or α-tubulin (a-tub)<b>.</b></p

SNX12 overexpression induces an accumulation of MVB-like structures and rescues the intralumenal vesicles that incorporate the EGF receptor.

<p>(<b>A-B</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. (<b>C</b>) Hrs (<b>D-E</b>) or SNX3 (<b>F-G</b>) was knocked down and mRFP-SNX12 (red) was overexpressed (<b>E-G</b>) or not (<b>D-F</b>). In each condition, HeLa cells were also transfected with GFP-Rab5^Q79L (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038949#pone.0038949-Pons1" target="_blank">[25]</a>. 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). (<b>D-G</b>) Representative pictures of experiments quantified in (<b>C</b>). GFP-Rab5^Q79L enlarged endosomes in each condition showed the EGFR localization after Hrs (<b>D-E</b>) or SNX3 (<b>F-G</b>) was knocked down and mRFP-SNX12 (red) was overexpressed (<b>E-G</b>) or not (<b>D-F</b>).</p

SNX12 overexpression inhibits EGFR transport and degradation without affecting retrograde and recycling transport routes.

<p>(<b>A-C</b>) After cell surface binding, EGF-biotin coupled to streptavidin^{AlexaFluor488} was endocytosed for 10 min (<b>A</b>) or 50 min (<b>B-C</b>) at 37°C in HeLa cells expressing mRFP1-SNX12. Cells were labeled with anti-EEA1 (<b>A</b>) or Lamp1 (<b>B-C</b>) antibodies and analyzed by triple channel fluorescence. (<b>D</b>) 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<b>.</b> (<b>E</b>) 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. (<b>F</b>) 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. (<b>A-C and E-F</b>) Scale bar indicates 10 µm.</p