Interchangeable but Essential Functions of SNX1 and SNX2 in the Association of Retromer with Endosomes and the Trafficking of Mannose 6-Phosphate Receptors

Abstract

The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cationindependent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function.

The retromer component SNX6 interacts with dynactin p150Glued and mediates endosome-to-TGN transport

“…Moreover, SNX5 was detected in immunoprecipitates of SNX1 and SNX2 ( Figure 2B), indicating that both SNX5 and SNX6 interact with SNX1/2. In sedimentation analyses of HeLa cell lysates on linear sucrose gradients, consistent with previous studies [15], SNX1, 2, 5 and 6 co-sedimented in lighter fractions, whereas Vps35 migrated in heavier fractions ( Figure 2C), suggesting that SNX6 and SNX1/2/5 form a subcomplex. Moreover, the peak fractions of the dynein/dynactin complex were distinct from those of the retromer subunits ( Figure 2C), suggesting that the motor-retromer association is weak or transient in vivo.…”

“…SNXs have diverse functions and are involved in a wide variety of physiological processes. SNX1 and SNX2 are essential for sorting and trafficking of transmembrane cargoes from endosomes to the trans-Golgi network (TGN) [15]. SNX3 binds to PI(3)P [16] and is required for biogenesis of endocytic carrier vesicles/multivesicular bodies [17].…”

“…Among its various cargoes are the Wnt chaperone Wntless, the auxin efflux carrier PIN in plants, and mannose-6-phosphate receptors (MPR) that are sorting receptors for acid hydrolases [20,21]. The retromer consists of two independently assembled subcomplexes, the cargo selective Vps26/29/35 trimer and the membrane-binding SNX dimer [15,22]. In yeast the latter subcomplex is formed by Vps5p and Vps17p [23,24], while in mammals the exact subunit composition of the SNX dimer is less clear.…”

“…In yeast the latter subcomplex is formed by Vps5p and Vps17p [23,24], while in mammals the exact subunit composition of the SNX dimer is less clear. SNX1 and SNX2 are mammalian orthologs of Vps5p [15,25], whereas there is no obvious sequence homolog of Vps17p in mammals. Most recently, an RNAi loss-of-function screen in HeLa cells identified SNX5 and SNX6 as putative components of the SNX subcomplex [26].…”

“…In vitro and in vivo studies have established that SNX1's PX domain binds to the endosome-enriched PI(3)P, and that its BAR domain enables the protein to bind curved membranes and drive membrane tubulation [27]. It is believed that SNX1 and SNX2 are interchangeable subunits of the SNX subcomplex of retromer [15], and that, through coincidence sensing of the high-curvature, PI(3)P-enriched microdomains of endosomal membranes by their PX and BAR domains, the SNXs, in the form of dimer or oligomer, generate and stabilize tubular structures from vacuolar portions of endosomes. These structures are thought to allow geometric sorting of transmembrane cargoes into tubulovesicular transport intermediates destined for TGN [27].…”