Determinants of the endosomal localization of sorting nexin 1

Abstract

The sorting nexin (SNX) family of proteins is characterized by sequence-related phox homology (PX) domains. A minority of PX domains bind with high affinity to phosphatidylinositol 3-phosphate [PI(3)P], whereas the majority of PX domains exhibit low affinity that is insufficient to target them to vesicles. SNX1 is located on endosomes, but its low affinity PX domain fails to localize in vivo. The NMR structure of the PX domain of SNX1 reveals an overall fold that is similar to high-affinity PX domains. However, the phosphatidylinositol (PI) binding pocket of the SNX1 PX domain is incomplete; regions of the pocket that are well defined in high-affinity PX domains are highly mobile in SNX1. Some of this mobility is lost upon binding PI(3)P. The C-terminal domain of SNX1 is a long helical dimer that localizes to vesicles but not to the early endosome antigen-1-containing vesicles where endogenous SNX1 resides. Thus, the obligate dimerization of SNX1 that is driven by the C-terminal domain creates a high-affinity PI binding species that properly targets the holo protein to endosomes.

Figure 1.

Distribution and binding specificities of PX domains. GFP-PX domains were expressed in E. coli, purified, and microinjected into HeLa cells without or with PI(3)P or PI(3,5)P2. (A) SNX3. (B) SNX3 + PI(3)P. (C) SNX3 + PI(3,5)P2. (D) PX domain of CISK. (E) PX domain of CISK + PI(3)P. (F) PX domain of CISK + PI(3,5)P2. (G) PX domain of SNX1. (H) PX domain of SNX2. (I) Cells were treated with the PI 3-kinase inhibitor LY294002 (50 μM) for 1 h before microinjection of GFP-CISK.

Figure 2.

Solution structure of the SNX1 PX domain. (A) Superposition of the backbone atoms of 10 refined solution structures for the SNX1-PX domain, highlighting in the lower part of the representation, the relatively low definition of the proline-rich loop connecting helices 1 and 2. (B) A ribbon representation of the lowest energy member of the ensemble shown at top, illustrating the topology within the tertiary fold of the SNX1-PX domain, the nomenclature of the secondary structure elements, and the location of residues predicted to be involved in phosphoinositide binding. The β-strands are yellow, and the α helices are green and blue. The proline-rich loop (labeled Pro-rich) is red except for the region of high mobility (orange). The β1-β2 loop is also highly mobile and orange.

Figure 3.

Phosphoinositide binding region. Superimposition of the PX domains of SNX1 (blue backbone, cyan side chains) and p40^phox (red backbone, orange side chains), focusing on the phosphoinositide binding region. The phosphoinositide bound to p40^phox is shown in green. The PX domain of p40^phox is representative of the high-affinity class of PX domains, all of which overlay closely. Residues proposed to be important for phosphoinositide binding in p40^phox, and their counterparts in SNX1, are labeled (where joint labeling occurs, the label for SNX1 is first).

Figure 4.

Colocalization of SNX1 with EEA1-positive endosomes. (A and B) COS 7 cells were fixed, permeabilized, and stained with goat anti-SNX1 or anti-SNX2 and mouse monoclonal anti-EEA1 antibodies. Detection was via donkey anti-goat and goat anti-mouse secondary antibodies conjugated to Alexa Fluor 594 and488. (C and D) Merged images of A and B. Inset is a close-up of the merged image of the boxed area (D).

Figure 5.

Endosomal colocalization of GFP-SNX1 and GFP-SNX2 with EEA1. HEK293 cells containing a chromosomally integrated copy of GFP-SNX1 or GFP-SNX2 were treated with 1 μg/ml tetracycline for 12 h to induce protein expression. Cells were fixed, permeabilized, and EEA1 was detected using mouse monoclonal anti-EEA1 and visualized using goat anti-mouse IgG H + L chains conjugated to Alexa Fluor 350. Cells were examined by deconvolution microscopy. Insets in C and F are close-ups of the boxed areas. Arrows show localization of GFP-SNX1 or GFP-SNX2 to microdomains of endosomes that are distinct from EEA1.

Figure 6.

Comparison of the localization of SNX1 with SNX2, SNX3, and CISK. The vesicular localization of endogenous SNX1 was compared with the vesicular localization of transfected GFP-SNX2, GFP-SNX3, and GFP-CISK by using immunofluorescent deconvolution microscopy. Endogenous SNX1 plus GFP-SNX2. Endogenous SNX1 plus GFP-SNX3. Endogenous SNX1 plus GFP-CISK. Endogenous EEA1 plus GFP-SNX3 and GFP-CISK. Panels on the right are merged images, and insets are higher magnification of the boxed areas.

Figure 7.

Vesicular localization of dimeric SNX1 PX domains. (A) GFP-C terminal domains of SNX1 (aa 272–522) was transfected into COS 7 cells, and its localization was compared with EEA1 by using immunofluorescence microscopy. (B) Comparison of the localization of holo SNX1 with EEA1.