The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1

Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1–AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.Membrane trafficking in plants shares many fundamental features with those in yeast and animals (Bassham et al., 2008). In general, vacuolar proteins are synthesized on the rough endoplasmic reticulum and then transported to vacuoles via the Golgi apparatus (Xiang et al., 2013; Robinson and Pimpl, 2014). The vacuolar trafficking in plants has been studied by monitoring the transport of reporter proteins to lytic vacuoles in vegetative cells and tissues (Jin et al., 2001; Pimpl et al., 2003; Miao et al., 2008; Niemes et al., 2010). Recently, seed storage proteins became a model cargo for monitoring the transport of endogenous vacuolar proteins in plants (Shimada et al., 2003a; Sanmartín et al., 2007; Isono et al., 2010; Pourcher et al., 2010; Uemura et al., 2012; Shirakawa et al., 2014). During seed maturation, a large amount of storage proteins are synthesized and sorted to specialized vacuoles, the protein storage vacuoles (PSVs). To properly deliver vacuolar proteins, sorting receptors play a critical role in recognizing the vacuole-targeting signal of the proteins. VACUOLAR PROTEIN SORTING10 and Man-6-P receptor function as sorting receptors for vacuolar/lysosomal proteins in the trans-Golgi network (TGN) of yeast and mammals, respectively. The best-characterized sorting receptors in plants are VACUOLAR SORTING RECEPTOR (VSR) family proteins (De Marcos Lousa et al., 2012). VSRs have been shown to function in sorting both storage proteins to PSVs (Shimada et al., 2003a; Fuji et al., 2007) and lytic cargos to lytic vacuoles (Zouhar et al., 2010).To sort the receptors in the TGN into vacuoles/lysosomes, the adaptor protein (AP) complex binds the cytosolic domain of the receptors. The AP complexes form evolutionarily conserved machinery that mediates the post-Golgi trafficking in eukaryotic cells (Robinson, 2004). There are five types of AP complexes, AP-1 to AP-5. The functions of AP-1, AP-2, and AP-3 have been established. AP-1 appears to be involved in trafficking between the TGN and endosomes (Hirst et al., 2012), AP-2 is involved in clathrin-mediated endocytosis (McMahon and Boucrot, 2011), and AP-3 is involved in protein trafficking from the TGN/endosomes to the vacuole/lysosomes (Dell’Angelica, 2009). However, little is known about AP-4 and AP-5. Mammalian AP-4 may be involved in basolateral sorting in polarized cells and in the transport of specific cargo proteins, such as the amyloid precursor protein APP, from the TGN to endosomes (Burgos et al., 2010). The fifth AP complex, AP-5, was recently identified, and its orthologs are widely conserved in the eukaryotic genomes (Hirst et al., 2011). The AP complexes exist as heterotetrameric proteins that consist of two large subunits (β1-5 and one each of ɣ/α/δ/ε/ζ), one medium subunit (µ1-5), and one small subunit (σ1-5). The sorting mechanism is best characterized for the medium (µ) subunit, which is known to recognize the Tyr-based YXXФ motif (where Ф represents Leu, Ile, Phe, Met, or Val) that is present in the cytosolic domains of cargo proteins (Ohno et al., 1995). Mutations of the YXXФ motif abolish the interaction with µ and alter the subcellular localization of the cargo proteins.The genome of Arabidopsis (Arabidopsis thaliana) contains all five sets of putative AP genes (Bassham et al., 2008; Hirst et al., 2011). The function of AP-4 in membrane trafficking and its physiological roles in plants are largely unknown. In this study, we identified and characterized the AP-4 complex in Arabidopsis. Mutants lacking the AP-4 subunits exhibited defects in VSR1-mediated vacuolar sorting of storage proteins in seeds. Our results provide new insights into the receptor-mediated vacuolar trafficking in post-Golgi pathways.