A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.Endocytosis mediates the internalization of cell-surface proteins and lipids in small vesicles that bud from the plasma membrane and deliver their cargo to endosomes (1). Once cargo proteins reach the endosomes, one important pathway they may follow is retrograde recycling, in which cargos are delivered from endosomes to the trans-Golgi network (TGN) (2). Many important membrane proteins, including signaling receptors and small molecular transporters, require retrograde recycling (2). Some well-studied examples include the cation-independent mannose 6-phosphate receptor, insulin-stimulated glucose transporter Glut4, and Wls/MIG-14, a protein that ferries Wnt ligands to the cell surface during their secretion (2, 3). Certain toxins and viruses co-opt such retrograde transport pathways during their toxic/infectious cycles. These include the bacterial toxins Shiga and cholera, plant exotoxins ricin and abrin, as well as adeno-associated virus type 5 (AAV5) and HIV-1 (2, 3).Relatively few studies have focused on how such recycling pathways function in polarized epithelial cells, although polarized epithelia are a very abundant and important cell type in the human body. The intestine of the nematode Caenorhabditis elegans is a powerful model system for the study of endocytic recycling in the context of polarized epithelial cells, and can be studied within their normal context of the intact living animal (4, 5). The C. elegans intestine is a simple epithelial tube composed of 20 cells arranged mostly in pairs (6). Like mammalian intestinal epithelial cells, those of the C. elegans intestine display readily apparent apicobasal polarity, with basolateral and apical domains separated by apical junctions (6). The intestinal luminal (apical) membranes display a dense microvillar brush border with an overlying glycocalyx and subapical terminal web (6). The basolateral membrane faces the body cavity, exchanging molecules between the intestine and peripheral tissues.We previously established several model transmembrane cargo markers for the analysis of basolateral endocytosis and recycling in the C. elegans intestine (5, 7–10). These include MIG-14-GFP, hTfR-GFP, and hTAC-GFP (5). MIG-14 (Wntless) and hTfR (human transferrin receptor) enter cells via clathrin-dependent endocytosis (5, 7, 11). hTAC (human IL-2 receptor α-chain) enters cells via clathrin-independent endocytosis (4, 5). hTfR and hTAC recycle to the plasma membrane via recycling endosomes, also known as the endocytic recycling compartment (4, 7). MIG-14 recycles to the TGN, but previous work had not tested whether MIG-14 transits the recycling endosome en route to the Golgi (12–14).Many studies in cultured cell lines indicate that there are multiple routes to the Golgi from endosomes, including proposed routes from the recycling endosome, in addition to the more commonly discussed early endosome and late endosome routes (15–19). For instance, CHO cell pulse–chase analysis of fusion proteins bearing the transmembrane and intracellular domains of retrograde recycling proteins, TGN38 and Furin, showed that TGN38 trafficked from the early endosome to recycling endosome to the Golgi, whereas Furin recycling involved transit from the early endosome to the late endosome to the Golgi (20, 21). TGN38 and Shiga toxin have been shown to require distinct sets of SNARE proteins to complete transport to the Golgi, also indicating that different cargos recycle to the Golgi in different types of vesicles (22). In addition, TGN38 requires recycling endosome regulator Rab11 and its effector FIP1/RCP for retrograde recycling, further indicating that the recycling endosome pathway is important in TGN38 retrieval to the Golgi (23). The recycling endosome regulator and dynamin superfamily-like ATPase EHD1/mRme-1 is also required for transport of several cargos from recycling endosomes to the Golgi (24–26). The cation-independent mannose 6-phosphate receptor has also been reported to require transport through the recycling endosome to reach the TGN (24, 25).Previous whole-genome analysis of genes required for yolk protein endocytosis in the C. elegans oocyte, a process that requires yolk receptor recycling, identified the Rho-family GTPase CDC-42 and its associated proteins PAR-3 and PAR-6 (PDZ domain proteins), as well as the C. elegans homolog of atypical protein kinase C, PKC-3 (27). Together, these proteins are often referred to as the anterior PAR complex, because they function together to establish and maintain anterior–posterior polarity in the early C. elegans embryo (28). This work showed that CDC-42 is enriched on RME-1–positive recycling endosomes in nonpolarized C. elegans coelomocytes and cultured mammalian fibroblasts (27). These and other data implicated the CDC-42/PAR complex in recycling endosome function, but further mechanistic insight was lacking (27, 29, 30). Other work showed that CDC-42–associated Bar-domain proteins TOCA-1 and TOCA-2 function redundantly in yolk endocytosis, also probably functioning at a postendocytic transport step (31).To better understand the function of the TOCA proteins, and potentially the anterior PAR complex, in membrane transport, we set out to analyze their function in the C. elegans intestine using the molecular tools that we had established in this tissue. Unlike the general recycling regulator RME-1, which affects all recycling cargo that we have tested in the C. elegans intestine, we found that toca-1; toca-2 double mutants strongly affected MIG-14 but not hTAC or hTfR. Further analysis connected TOCA-1 and TOCA-2 to the CDC-42/PAR complex in this process, and further indicated that these proteins function with WVE-1, a core subunit of the actin nucleation complex WAVE. These results indicated a requirement for TOCA/CDC-42/PAR/WAVE in retrograde recycling, in addition to the well-known retromer complex, which contains a similar complement of molecules implicated in membrane binding, membrane bending, and actin nucleation. Finally, we established the C. elegans homolog of retrograde recycling cargo TGN38 (TGN-38) as a retrograde recycling cargo in the intestine. Experiments with GFP-tagged TGN-38 allowed us to compare the cargo transport blocks imposed by depletion of a subunit of the retromer complex (SNX-3) with the block imposed by depletion of CDC-42, as a representative of the TOCA/CDC-42/PAR/WAVE group (32). These experiments indicated a specific block at the early endosome after retromer depletion, whereas CDC-42 depletion blocked TGN-38 retrograde recycling most strongly at the recycling endosome. Taken together, these results demonstrate an important role for TOCA/CDC-42/PAR/WAVE acting at the recycling endosome after retromer function at the early endosome. We also provide evidence for the physiological importance of this pathway in WNT signaling, focusing on the polarity of the ALM mechanosensory neurons.