Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval

Journal of Cell Science, Jul 2007

Matthew N. J. Seaman

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Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval

Matthew N. J. Seaman 0 0 University of Cambridge, Cambridge Institute for Medical Research/Clinical Biochemistry, Wellcome Trust/MRC building, Addenbrookes Hospital , Cambridge, CB2 0XY , UK - Summary The cation-independent mannose 6-phosphate receptor (CIMPR) cycles between the trans-Golgi network (TGN) and endosomes to mediate sorting of lysosomal hydrolases. The endosome-to-TGN retrieval of the CIMPR requires the retromer complex. Genetic, biochemical and structural data support the hypothesis that the retromer can directly bind to the tail of the CIMPR, to sort the CIMPR into ce vesicles and tubules for retrieval to the TGN. Presently, n however, no known retromer sorting motif in the tail of the e ic CIMPR has been identified. Using CD8-reporter proteins S carrying the cytoplasmic tail of the CIMPR we have l l e C f o l Introduction a n The function of the different organelles that comprise the ru secretory and endocytic pathways in eukaryotic cells is o determined by the complement of resident proteins present J within the respective organelle. The mechanisms that govern membrane protein localisation to the different organelles rely on intrinsic information in the membrane protein such as sorting motifs in the cytoplasmic domain and extrinsic factors such as coat-proteins that recognise the sorting motifs. A well-studied example of these concepts is the sorting of membrane proteins by the clathrin-coated vesicle (CCV) adaptor proteins through recognition of the YXX sorting motif. This tetra-peptide motif, comprising Tyr-X-X (where X is any amino acid)-bulky hydrophobic, is bound by the medium chain ( ) subunit of adaptor complexes resulting in the protein containing the YXX motif being concentrated in CCVs (Ohno et al., 1995; Owen and Evans, 1998) (reviewed in Bonifacino and Traub, 2003). Another example of sorting motifs interacting with coat proteins is the recognition of acidic di-leucine motifs by Golgi-associated ear-containing ARF-binding (GGA) proteins. GGA proteins bind to the consensus sequence, Asp-X-X-Leu-Leu (DXXLL) and function at the trans-Golgi network (TGN) to direct specific cargo proteins into CCVs (Puertollano et al., 2001; Misra et al., 2002). Sorting motifs, therefore, are often just two to four amino acids present in the correct molecular context. YXX motifs must be at least seven amino acids from the transmembrane domain (Rohrer et al., 1996) whereas acidic di-leucine motifs are usually near the end of systematically dissected the CIMPR tail to identify a novel, conserved aromatic-containing sorting motif that is critical for the endosome-to-TGN retrieval of the CIMPR and for the interaction with retromer and the clathrin adaptor AP-1. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/14/2378/DC1 the cytoplasmic domain of proteins that traffic between the TGN and endosomes. Membrane proteins that traffic in the post-Golgi endomembrane system often contain multiple sorting motifs that function to direct the transport of the membrane protein between the various compartments such as the TGN, endosomes and the plasma membrane. A good example of this is the cation-independent mannose 6-phosphate receptor (CIMPR) which binds to lysosomal hydrolases in the TGN and is sorted into CCVs by GGA proteins in concert with the AP-1 clathrin adaptor (Ghosh et al., 2003; Doray et al., 2002). The cytoplasmic tail of the CIMPR, therefore, has both YXX and DXXLL motifs. In the case of the CIMPR, the affinity of the GGAs for DXXLL motifs is augmented by casein kinase-mediated phosphorylation of a serine residue that immediately precedes the aspartate of the acidic dileucine (Kato et al., 2002). Following the delivery of the CIMPR to an endosome, the CIMPR releases its ligand and is then recycled back to the TGN to undergo further rounds of hydrolase sorting. Retrieval of the CIMPR from the endosome to the TGN is mediated by the retromer complex (Seaman, 2004; Arighi et al., 2004; Carlton et al., 2004) and has also been shown to require the function of the TIP47 and PACS1 proteins (reviewed by Seaman, 2005; Bonifacino and Rojas, 2006). Retromer was first described in yeast and is required for the endosome-to-Golgi retrieval of the vacuolar hydrolase receptor Vps10p, the yeast functional equivalent of the CIMPR (Seaman et al., 1997; Seaman et al., 1998). Retromer comprises five proteins in yeast, Vps35p, Vps29p, Vps26p, Vps5p and Vps17p, which are conserved in mammals (with the exception of Vps17p that has no clear homologue) (reviewed by Seaman, 2005). Retromer can recognise cargo such as Vps10p and the CIMPR through interactions with Vps35p or mammalian VPS35, respectively (Nothwehr et al., 2000; Arighi et al., 2004). Retromer can also reshape the membrane by the action of the sorting nexin components Vps5p and its mammalian homologue, sorting nexin-1 (SNX1) (Seaman et al., 1998; Carlton et al., 2004). Recent structural and biochemical studies of VPS29 h (...truncated)


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Matthew N. J. Seaman. Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval, Journal of Cell Science, 2007, pp. 2378-2389, 120/14, DOI: 10.1242/jcs.009654