An Essential Role for SNX1 in Lysosomal Sorting of Protease-activated Receptor-1: Evidence for Retromer-, Hrs-, and Tsg101-independent Functions of Sorting Nexins

Abstract

Sorting nexin 1 (SNX1) and SNX2 are the mammalian homologues of the yeast Vps5p retromer component that functions in endosome-to-Golgi trafficking. SNX1 is also implicated in endosome-to-lysosome sorting of cell surface receptors, although its requirement in this process remains to be determined. To assess SNX1 function in endocytic sorting of protease-activated receptor-1 (PAR1), we used siRNA to deplete HeLa cells of endogenous SNX1 protein. PAR1, a G-protein-coupled receptor, is proteolytically activated by thrombin, internalized, sorted predominantly to lysosomes, and efficiently degraded. Strikingly, depletion of endogenous SNX1 by siRNA markedly inhibited agonist-induced PAR1 degradation, whereas expression of a SNX1 siRNA-resistant mutant protein restored agonist-promoted PAR1 degradation in cells lacking endogenous SNX1, indicating that SNX1 is necessary for lysosomal degradation of PAR1. SNX1 is known to interact with components of the mammalian retromer complex and Hrs, an early endosomal membrane-associated protein. However, activated PAR1 degradation was not affected in cells depleted of retromer Vps26/Vps35 subunits, Hrs or Tsg101, an Hrs-interacting protein. We further show that SNX2, which dimerizes with SNX1, is not essential for lysosomal sorting of PAR1, but rather can regulate PAR1 degradation by disrupting endosomal localization of endogenous SNX1 when ectopically expressed. Together, our findings establish an essential role for endogenous SNX1 in sorting activated PAR1 to a distinct lysosomal degradative pathway that is independent of retromer, Hrs, and Tsg101.

The retromer component sorting nexin-1 is required for efficient retrograde transport of Shiga toxin from early endosome to the trans Golgi network

“…Like Shiga and cholera toxins, ricin is an ERtrafficking toxin; but unlike STxB and CI-MPR, its endosome-to-TGN transport does not appear to depend on clathrin or Rab9 (Lombardi et al, 1993;Iversen et al, 2001;Lauvrak et al, 2004;Saint-Pol et al, 2004). The finding that SNX2 and SNX1 appear to have independent properties with regards to toxin traffic (Skanland et al, 2007) (and this study), but also with respect to receptor sorting (Gullapalli et al, 2004;Gullapalli et al, 2006), point towards a model in which SNX1 and SNX2 can function independently of each other and, at least for SNX1, independently of Vps26 (Gullapalli et al, 2006). In support of this model is the result from a recent RNAi loss-of-function screen that identified sorting nexins SNX5 and SNX6 as additional candidates for the retromer complex as their suppression induces a 'retromer-like phenotype' with regard to CI-MPR redistribution (Wassmer et al, 2006).…”

Visualisation of macropinosome maturation by the recruitment of sorting nexins

“…For this to be the case, 'unwanted' components would need to be sorted and segregated from those targeted to the lysosome. The sorting nexins, SNX1 and SNX5, represent promising candidates for this role in the macropinosome as they have both been implicated in the sorting and trafficking of a number of different cargo proteins (Gullapalli et al, 2006;Kurten et al, 1996;Otsuki et al, 1999;Reuter et al, 2003). By using a tubular element to remove plasma membrane components rather than a spherical vesicle, the ratio of surface area to volume in the macropinosome is reduced, effectively 'tightening the slack' on the compartment.…”

Evolutionary reconstruction of the retromer complex and its function in Trypanosoma brucei

“…In addition, Table S2. Protein name abbreviations and selected references relating to their interaction with retromer are as follows: Vps10, vacuolar protein sorting 10, similar to mammalian sortilins (Seaman et al, 1997); EHD1, Eps15-homology-domain-containing protein-1 (Gokool et al, 2007); STE13 (DPAP), dipeptydyl aminopeptidase (Nothwehr et al, 1999;Nothwehr et al, 2000); KEX2, killer expression defective 2 serine protease (Nothwehr and Hindes, 1997); FET3, ferrous transport 3 (Strochlic et al, 2007); FTR1, Fe transporter 1, forms part of the yeast reductive iron transporter with Fet3 (Strochlic et al, 2007), PEP12, carboxypeptidase-Ydeficient 12 (Hettema et al, 2003); CI-MPR, cation-independent mannose 6-phosphate receptor (Arighi et al, 2004;Seaman, 2004); VSR1, vacuolar sorting receptor 1 (Yamazaki et al, 2008); BACE, -secretase, also known as memapsin 2 (He et al, 2005); LRP6, lipoprotein-receptor-related protein 6 (George et al, 2007); PAR1, protease-activated receptor-1 (Gullapalli et al, 2006); PIGR, polymeric immunoglobulin receptor (Verges et al, 2004;Verges et al, 2007); EGFR, epidermal growth factor receptor (Gullapalli et al, 2004); wntless, G-protein-coupled receptor 177 or Wnt receptor (Eaton, 2008); BTN2, Batten disease protein 2 (Kama et al, 2007). Also included are results of searches for GNPTAB and NAGPA, enzymes required for synthesis of mannose 6-phosphate (boxed).…”