Glycosylphosphatidylinositol (GPI) Modification Serves as a Primary Plasmodesmal Sorting Signal

Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communication in plants. The Pd-associated β-1,3-glucanase (BG_pap) and CALLOSE BINDING PROTEIN1 (PDCB1) were identified as key regulators of Pd conductivity. Both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs) carrying a conserved GPI modification signal. However, the subcellular targeting mechanism of these proteins is unknown, particularly in the context of other GPI-APs not associated with Pd. Here, we conducted a comparative analysis of the subcellular targeting of the two Pd-resident and two unrelated non-PdGPI-APs in Arabidopsis (Arabidopsisthaliana). We show that GPI modification is necessary and sufficient for delivering both BG_pap and PDCB1 to Pd. Moreover, the GPI modification signal from both Pd- and non-PdGPI-APs is able to target a reporter protein to Pd, likely to plasma membrane microdomains enriched at Pd. As such, the GPI modification serves as a primary Pd sorting signal in plant cells. Interestingly, the ectodomain, a region that carries the functional domain in GPI-APs, in Pd-resident proteins further enhances Pd accumulation. However, in non-PdGPI-APs, the ectodomain overrides the Pd targeting function of the GPI signal and determines a specific GPI-dependent non-Pd localization of these proteins at the plasma membrane and cell wall. Domain-swap analysis showed that the non-Pd localization is also dominant over the Pd-enhancing function mediated by a Pd ectodomain. In conclusion, our results indicate that segregation between Pd- and non-PdGPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.Plant cells are interconnected with cross-wall membranous channels called plasmodesmata (Pd). Recent studies have shown that the region of the plasma membrane (PM) lining the Pd channel is a specialized membrane microdomain whose lipid and protein composition differs from the rest of the PM (Tilsner et al., 2011, 2016; Bayer et al., 2014; González-Solís et al., 2014; Grison et al., 2015). In a similar manner, the cell wall domain surrounding the Pd channel is specialized and, unlike the rest of the cell wall, is devoid of cellulose, rich in pectin, and contains callose (an insoluble β-1,3-glucan; Zavaliev et al., 2011; Knox and Benitez-Alfonso, 2014). In response to physiological signals, callose can be transiently deposited and degraded at Pd, which provides a mechanism for controlling the Pd aperture in diverse developmental and stress-related processes (Zavaliev et al., 2011). Control of Pd functioning is mediated by proteins that are specifically targeted to Pd. Plasmodesmal proteins localized to the PM domain of Pd can be integral transmembrane proteins, such as Pd-localized proteins (Thomas et al., 2008), the receptor kinase ARABIDOPSIS CRINKLY4 (Stahl et al., 2013), and callose synthases (Vatén et al., 2011). Alternatively, Pd proteins can associate with the membrane through a lipid modification like myristoylation (e.g. remorins; Raffaele et al., 2009) or be attached by a glycosylphosphatidylinositol (GPI) anchor (e.g. Pd-associated β-1,3-glucanases [BG_pap]; Levy et al., 2007; Rinne et al., 2011; Benitez-Alfonso et al., 2013), Pd-associated callose-binding proteins (PDCBs; Simpson et al., 2009), and LYSIN MOTIF DOMAIN-CONTAINING PROTEIN2 (LYM2; Faulkner et al., 2013).Among the known Pd proteins involved in Pd-specific callose degradation is BG_pap, a cell wall enzyme carrying a glycosyl hydrolase family 17 (GH17) module as its functional domain (Levy et al., 2007). Another group of proteins controlling callose dynamics at Pd are PDCBs that harbor a callose-binding domain termed carbohydrate-binding module 43 (CBM43), implicated in stabilizing callose at Pd (Simpson et al., 2009). Some β-1,3-glucanases may combine the two callose-modifying activities by harboring both GH17 and CBM43 functional domains, and several such proteins were shown to localize to Pd (Rinne et al., 2011; Benitez-Alfonso et al., 2013; Gaudioso-Pedraza and Benitez-Alfonso, 2014). A distinct feature of BG_pap and PDCBs is that both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs). The GPI anchor is a form of posttranslational modification common to many cell surface proteins in all eukaryotes. GPI-APs are covalently attached to the outer leaflet of the PM through the GPI anchor. The basic structure of the anchor consists of ethanolamine phosphate, followed by a glycan chain of three Man residues and glucosamine, followed by phosphatidylinositol lipid moiety (EtNP-6Manα1-2Manα1-6Manα1-4GlcNα1-6myoinositol-1-P-lipid; Ferguson et al., 2009). All predicted GPI-APs carry an N-terminal secretion signal peptide (SP) similar to other secreted proteins. Distinctly, GPI-APs also carry a structurally conserved 25- to 30-residue C-terminal GPI attachment signal, which typically begins with a small amino acid (e.g. Ala, Asn, Asp, Cys, Gly, or Ser) termed omega, followed by a spacer region of five to 10 polar residues, and ending with a transmembrane segment of 15 to 20 hydrophobic residues (Ferguson et al., 2009). The entire region between the N-terminal and the C-terminal signals of a GPI-AP is termed the ectodomain and carries the protein’s functional domain(s). The GPI modification process takes place in the lumenal face of the endoplasmic reticulum (ER) in a cotranslational manner. Upon translocation into the ER, a GPI-AP is stabilized in the ER membrane by its C-terminal signal, which is concurrently cleaved after the omega amino acid, and a preassembled GPI anchor is covalently attached to the C terminus of the omega amino acid. After attachment to a protein, the GPI anchor undergoes a series of modifications (remodeling), both at the glucan chain and at the lipid moiety. Such remodeling is crucial for the sorting of GPI-APs in the secretory pathway and the subsequent lateral heterogeneity at the PM (Kinoshita, 2015). In particular, the addition of saturated fatty acid chains to the lipid moiety of the anchor leads to the enriched accumulation of GPI-APs in the PM microdomains, also termed lipid rafts (Muñiz and Zurzolo, 2014). In Arabidopsis (Arabidopsisthaliana), GPI modification has been predicted for 210 proteins of diverse functions at the PM or the cell wall or both (Borner et al., 2002). Despite extensive research on the GPI modification pathway and the function of GPI-APs in mammalian and yeast cells, such knowledge in plant systems is scarce. In particular, despite an emerging role of GPI-APs in the regulation of the cell wall domain of Pd, their subcellular targeting and compartmentalization mechanism have not been studied. In addition, it is not known how the targeting mechanism of Pd-resident GPI-APs is different from that of other classes of GPI-APs, which are not localized to Pd.In this study, we investigated the subcellular targeting mechanism of Pd-associated callose-modifying GPI-APs, BG_pap and PDCB1, and compared it with that of two unrelated non-PdGPI-APs, ARABINOGALACTAN PROTEIN4 (AGP4) and LIPID TRANSFER PROTEIN1 (LTPG1). Using sequential fluorescent labeling of protein domains, we found that the C-terminal GPI modification signal present in both Pd- and non-Pd GPI-APs can function as a primary signal in targeting proteins to the Pd-enriched PM domain. Moreover, we show that while the GPI signal is sufficient for Pd targeting, the ectodomains in BG_pap and PDCB1 further enhance their accumulation at Pd. In contrast, the ectodomains in non-PdGPI-APs mediate exclusion of the proteins from the Pd-enriched targeting pathway. The Pd exclusion effect was found to be dominant over the Pd-targeting function of the GPI signal and the Pd-enhancing function of the Pd ectodomain, and it possibly occurs prior to PM localization. Our findings thus uncover a novel Pd-targeting signal and provide, to our knowledge, the first evidence of the cellular mechanism that regulates the sorting of GPI-APs in plants.