A dynamic partitioning mechanism polarizes membrane protein distribution.

The plasma membrane is widely regarded as the hub of the numerous signal transduction activities. Yet, the fundamental biophysical mechanisms that spatiotemporally compartmentalize different classes of membrane proteins remain unclear. Using multimodal live-cell imaging, here we first show that several lipid-anchored membrane proteins are consistently depleted from the membrane regions where the Ras/PI3K/Akt/F-actin network is activated. The dynamic polarization of these proteins does not depend upon the F-actin-based cytoskeletal structures, recurring shuttling between membrane and cytosol, or directed vesicular trafficking. Photoconversion microscopy and single-molecule measurements demonstrate that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane which enable their selective segregation. When these diffusion coefficients are incorporated into an excitable network-based stochastic reaction-diffusion model, simulations reveal that the altered affinity mediated selective partitioning is sufficient to drive familiar propagating wave patterns. Furthermore, normally uniform integral and lipid-anchored membrane proteins partition successfully when membrane domain-specific peptides are optogenetically recruited to them. We propose "dynamic partitioning" as a new mechanism that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins during various physiological processes where membrane polarizes.

p39-associated Cdk5 activity regulates dendritic morphogenesis.

Dendrites, branched structures extending from neuronal cell soma, are specialized for processing information from other neurons. The morphogenesis of dendritic structures is spatiotemporally regulated by well-orchestrated signaling cascades. Dysregulation of these processes impacts the wiring of neuronal circuit and efficacy of neurotransmission, which contribute to the pathogeneses of neurological disorders. While Cdk5 (cyclin-dependent kinase 5) plays a critical role in neuronal dendritic development, its underlying molecular control is not fully understood. In this study, we show that p39, one of the two neuronal Cdk5 activators, is a key regulator of dendritic morphogenesis. Pyramidal neurons deficient in p39 exhibit aberrant dendritic morphology characterized by shorter length and reduced arborization, which is comparable to dendrites in Cdk5-deficient neurons. RNA sequencing analysis shows that the adaptor protein, WDFY1 (WD repeat and FYVE domain-containing 1), acts downstream of Cdk5/p39 to regulate dendritic morphogenesis. While WDFY1 is elevated in p39-deficient neurons, suppressing its expression rescues the impaired dendritic arborization. Further phosphoproteomic analysis suggests that Cdk5/p39 mediates dendritic morphogenesis by modulating various downstream signaling pathways, including PI3K/Akt-, cAMP-, or small GTPase-mediated signaling transduction pathways, thereby regulating cytoskeletal organization, protein synthesis, and protein trafficking.

An Improved Intracellular Synthetic Lipidation-Induced Plasma Membrane Anchoring System for SNAP-Tag Fusion Proteins.

The ability to chemically introduce lipid modifications to specific intracellular protein targets would enable the conditional control of protein localization and activity in living cells. We recently developed a chemical-genetic approach in which an engineered SNAP-tag fusion protein can be rapidly relocated and anchored from the cytoplasm to the plasma membrane (PM) upon post-translational covalent lipopeptide conjugation in cells. However, the first-generation system achieved only low to moderate protein anchoring (recruiting) efficiencies and lacked wide applicability. Herein, we describe the rational design of an improved system for intracellular synthetic lipidation-induced PM anchoring of SNAP-tag fusion proteins. In the new system, the SNAPf protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAPΔ, is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl-DCys lipopeptidomimetic, called mDcBCP, is used as a cell-permeable chemical probe for intracellular SNAP-tag lipidation. The use of this combination allows significantly improved conditional PM anchoring of SNAP-tag fusion proteins. This second-generation system was applied to activate various signaling proteins, including Tiam1, cRaf, PI3K, and Sos, upon synthetic lipidation-induced PM anchoring/recruitment, offering a new and useful research tool in chemical biology and synthetic biology.

Cell-Free Protein Translation System for Expression of Lipid-Binding Proteins Tagged with Small epitopes and Their Use in Protein-Lipid Overlay Assays.

We adapted an efficient cell-free protein synthesis-based protocol for the production of lipid-binding proteins. The experimental procedures are based on the following steps: (1) cell-free synthesis of soluble, lipid-binding proteins fused to small tags; (2) analysis by dot blot of the accessibility of antibodies to the small tags. (3) protein lipid overlay assay with, immunodetection of bound protein by either chemiluminescence or fluorescence. We also provide a fast and inexpensive protocol for homemade lipid nitrocellulose strips spotted with acidic lipids (mostly phosphoinositides) extracted from plant tissues. These homemade lipid strips can be used for preliminary screen and characterization of putative phosphoinositide-binding proteins.

Membrane insertion and intercellular transfer of glycosylphosphatidylinositol-anchored proteins: potential therapeutic applications.

Anchorage of a subset of cell surface proteins in eukaryotic cells is mediated by a glycosylphosphatidylinositol (GPI) moiety covalently attached to the carboxy-terminus of the protein moiety. Experimental evidence for the potential of GPI-anchored proteins (GPI-AP) of being released from cells into the extracellular environment has been accumulating, which involves either the loss or retention of the GPI anchor. Release of GPI-AP from donor cells may occur spontaneously or in response to endogenous or environmental signals. The experimental evidence for direct insertion of exogenous GPI-AP equipped with the complete anchor structure into the outer plasma membrane bilayer leaflets of acceptor cells is reviewed as well as the potential underlying molecular mechanisms. Furthermore, promiscuous transfer of certain GPI-AP between plasma membranes of different cells in vivo under certain (patho)physiological conditions has been reported. Engineering of target cell surfaces using chimeric GPI-AP with complete GPI anchor may be useful for therapeutic applications.

Scaffold protein Lin7 family in membrane skeletal protein complex in mouse seminiferous tubules.

The membrane skeletal complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell-cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.

A global analysis of IFT-A function reveals specialization for transport of membrane-associated proteins into cilia.

Intraflagellar transport (IFT), which is essential for the formation and function of cilia in most organisms, is the trafficking of IFT trains (i.e. assemblies of IFT particles) that carry cargo within the cilium. Defects in IFT cause several human diseases. IFT trains contain the complexes IFT-A and IFT-B. To dissect the functions of these complexes, we studied a Chlamydomonas mutant that is null for the IFT-A protein IFT140. The mutation had no effect on IFT-B but destabilized IFT-A, preventing flagella assembly. Therefore, IFT-A assembly requires IFT140. Truncated IFT140, which lacks the N-terminal WD repeats of the protein, partially rescued IFT and supported formation of half-length flagella that contained normal levels of IFT-B but greatly reduced amounts of IFT-A. The axonemes of these flagella had normal ultrastructure and, as investigated by SDS-PAGE, normal composition. However, composition of the flagellar 'membrane+matrix' was abnormal. Analysis of the latter fraction by mass spectrometry revealed decreases in small GTPases, lipid-anchored proteins and cell signaling proteins. Thus, IFT-A is specialized for the import of membrane-associated proteins. Abnormal levels of the latter are likely to account for the multiple phenotypes of patients with defects in IFT140.This article has an associated First Person interview with the first author of the paper.

The membrane palmitoylated protein, MPP6, is involved in myelin formation in the mouse peripheral nervous system.

A membrane skeletal molecular complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6)-Lin7-cell adhesion molecule 4 (CADM4), is incorporated in Schwann cells, especially in Schmidt-Lanterman incisures (SLIs), in the mouse peripheral nervous system (PNS). MPP6, Lin7, and CADM4 are transported to SLIs by 4.1G. In this study, we created MPP6-deficient mice and evaluated myelin structure and MPP6 protein complexes. In SLIs in MPP6-deficient nerves, Lin7 was rarely detected by immunohistochemistry and western blotting, but the localization and amount of CADM4 and 4.1G were not altered. Motor activity was not significantly impaired in a tail-suspension test, but the sciatic nerves of MPP6-deficient mice had thicker myelin in internodes by electron microscopy compared to that of wild-type mice. These results indicate that the MPP6-Lin7 complex regulates myelin formation.

Studying assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy.

Significant progress has been made in obtaining structural insight into the assembly of the ß-barrel assembly machinery complex (BAM). These crystallography and electron microscopy studies used detergent as a membrane mimetic and revealed structural variations in the central domain, BamA, as well as in the lipoprotein BamC. We have used cellular solid-state NMR spectroscopy to examine the entire BamABCDE complex in native outer membranes and obtained data on the BamCDE subcomplex in outer membranes, in addition to synthetic bilayers. To reduce spectral crowding, we utilized proton-detected experiments and employed amino-acid specific isotope-labelling in (13C, 13C) correlation experiments. Taken together, the results provide insight into the overall fold and assembly of the BAM complex in native membranes, in particular regarding the structural flexibility of BamC in the absence of the core unit BamA.

Generating anchors only to lose them: The unusual story of glycosylphosphatidylinositol anchor biosynthesis and remodeling in yeast and fungi.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present ubiquitously at the cell surface in all eukaryotes. They play a crucial role in the interaction of the cell with its external environment, allowing the cell to receive signals, respond to challenges, and mediate adhesion. In yeast and fungi, they also participate in the structural integrity of the cell wall and are often essential for survival. Roughly four decades after the discovery of the first GPI-APs, this review provides an overview of the insights gained from studies of the GPI biosynthetic pathway and the future challenges in the field. In particular, we focus on the biosynthetic pathway in Saccharomyces cerevisiae, which has for long been studied as a model organism. Where available, we also provide information about the GPI biosynthetic steps in other yeast/ fungi. Although the core structure of the GPI anchor is conserved across organisms, several variations are built into the biosynthetic pathway. The present Review specifically highlights these variations and their implications. There is growing evidence to suggest that several phenotypes are common to GPI deficiency and should be expected in GPI biosynthetic mutants. However, it appears that several phenotypes are unique to a specific step in the pathway and may even be species-specific. These could suggest the points at which the GPI biosynthetic pathway intersects with other important cellular pathways and could be points of regulation. They could be of particular significance in the study of pathogenic fungi and in identification of new and specific antifungal drugs/ drug targets. © 2018 IUBMB Life, 70(5):355-383, 2018.

Legionella effector AnkX interacts with host nuclear protein PLEKHN1.

BACKGROUND: The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires' disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes. The pathogen delivers over 300 effector proteins into the host cell, altering host pathways in a manner that sets the stage for efficient pathogen replication. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. However, the current understanding of AnkX's interaction with host proteins and the means through which it exerts its cellular function is limited. RESULTS: Here, we investigated the protein interaction network of AnkX by using the nucleic acid programmable protein array (NAPPA), a high-density platform comprising 10,000 unique human ORFs. This approach facilitated the discovery of PLEKHN1 as a novel interaction partner of AnkX. We confirmed this interaction through multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Structured illumination microscopy revealed that endogenous PLEKHN1 is found in the nucleus and on vesicular compartments, whereas ectopically produced AnkX co-localized with lipid rafts at the plasma membrane. In mammalian cells, HaloTag-AnkX co-localized with endogenous PLEKHN1 on vesicular compartments. A central fragment of AnkX (amino acids 491-809), containing eight ankyrin repeats, extensively co-localized with endogenous PLEKHN1, indicating that this region may harbor a new function. Further, we found that PLEKHN1 associated with multiple proteins involved in the inflammatory response. CONCLUSIONS: Altogether, our study provides evidence that in addition to Rab GTPases, the L. pneumophila effector AnkX targets nuclear host proteins and suggests that AnkX may have novel functions related to manipulating the inflammatory response.

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition.

Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "ß-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively but often requires non-physiological conditions and time scales and is strongly influenced by properties of the lipid bilayer, including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the ß-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel-phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA, and another model protein (OmpG) folded at significantly different rates and that an α-helix embedded inside the EspP ß-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding.

Sensor kinase KinB and its pathway-associated key factors sense the signal of nutrition starvation in sporulation of Bacillus subtilis.

Bacillus subtilis responds to environmental stress cues and develops endospores for survival. In the process of endospore formation, sporulation initiation is a vital stage and this stage is governed by autophosphorylation of the sensor histidine kinases. The second major sensor kinase KinB perceives the intracellular changes of GTP and ATP during sporulation. However, determination of the environmental signals as well as its related signaling pathway of KinB requires further elucidation. Our current study found that, contrary to the sporulation failure induced by ΔkinA in the nutrient-rich 2× SG medium, the sensor kinase KinB sensed the environmental cues in the nutrient-poor MM medium. Two other membrane proteins, KapB and KbaA, also responded similarly to the same external signal as KinB. Both KapB and KbaA acted upstream of KinB, but they exerted their regulation upon KinB independently. Furthermore, we demonstrated that both the SH3 domain and the α-helix structure in KapB are required for sensing or transducing the signal of sporulation initiation. Collectively, our work here supplied the direct evidences that KinB and its pathway sense the external signal of nutrient starvation in MM medium, and further analyzes the interrelationship among KinB, KbaA, and KapB.

Role of N-myristoylation in stability and subcellular localization of the CLPABP protein.

Cardiolipin and phosphatidic acid-binding protein (CLPABP) controls the stability of the mRNA harboring an AU-rich element (ARE) in the 3' UTR with the help of the RNA stabilizer, human antigen R (HuR). Although CLPABP is localized on the mitochondrial surface as a large protein-RNA complex, its precise role is not yet known. Recently, CLPABP was identified as an N-myristoylated protein. Here, we demonstrate the effects of N-myristoylation on the functions of CLPABP. In the present study, compared to the wild-type protein that possessed the "MG" motif at the N-terminus for N-myristoylation, the mutant CLPABP protein that lacked N-myristoylation modification site was unstable. Furthermore, the expression of the G/A mutant of CLPABP, which lacked N-myristoylation site, induced morphological alterations in mitochondria. Because pleckstrin homology domain-deleted mutant, which was fused with the N-myristoylation site derived from intact CLPABP, could not colocalize with mitochondria, N-myristoylation of CLPABP was predicted to affect its stability onto the mitochondrial membrane rather than its subcellular localization.

Incorporation of Artificial Lipid-Anchored Proteins into Cultured Mammalian Cells.

Exogenous lipid-anchored proteins can be incorporated into the plasma membranes of living mammalian cells, allowing the chemical structure of the incorporated protein and its lipid anchor to be controlled (and varied) to a much greater degree than is possible for proteins expressed by the cells themselves. This technology offers a variety of potential applications, including incorporating novel and complex protein constructs into cell surfaces and exploring structure-function relationships for biologically important lipid-anchored proteins such as glycosylphosphatidylinositol-anchored proteins. Here we describe detailed methods for stable incorporation of artificial lipid-anchored proteins into cultured mammalian cells under mild, nonperturbing conditions.

The ß-barrel assembly machinery in motion.

In Gram-negative bacteria, the biogenesis of ß-barrel outer membrane proteins (OMPs) is mediated by the ß-barrel assembly machinery (BAM) complex. During the past decade, structural and functional studies have collectively contributed to advancing our understanding of the structure and function of the BAM complex; however, the exact mechanism that is involved remains elusive. In this Progress article, we discuss recent structural studies that have revealed that the accessory proteins may regulate essential unprecedented conformational changes in the core component BamA during function. We also detail the mechanistic insights that have been gained from structural data, mutagenesis studies and molecular dynamics simulations, and explore two emerging models for the BAM-mediated biogenesis of OMPs in bacteria.

p39 Is Responsible for Increasing Cdk5 Activity during Postnatal Neuron Differentiation and Governs Neuronal Network Formation and Epileptic Responses.

Two distinct protein cofactors, p35 and p39, independently activate Cyclin-dependent kinase 5 (Cdk5), which plays diverse roles in normal brain function and the pathogenesis of many neurological diseases. The initial discovery that loss of p35 impairs neuronal migration in the embryonic brain prompted intensive research exploring the function of p35-dependent Cdk5 activity. In contrast, p39 expression is restricted to the postnatal brain and its function remains poorly understood. Despite the robustly increased Cdk5 activity during neuronal differentiation, which activator is responsible for enhancing Cdk5 activation and how the two distinct activators direct Cdk5 signaling to govern neuronal network formation and function still remains elusive. Here we report that p39, but not p35, is selectively upregulated by histone acetylation-mediated transcription, which underlies the robust increase of Cdk5 activity during rat and mouse neuronal differentiation. The loss of p39 attenuates overall Cdk5 activity in neurons and preferentially affects phosphorylation of specific Cdk5 targets, leading to aberrant axonal growth and impaired dendritic spine and synapse formation. In adult mouse brains, p39 deficiency results in dysregulation of p35 and Cdk5 targets in synapses. Moreover, in contrast to the proepileptic phenotype caused by the lack of p35, p39 loss leads to deficits in maintaining seizure activity and induction of immediate early genes that control hippocampal excitability. Together, our studies demonstrate essential roles of p39 in neuronal network development and function. Furthermore, our data support a model in which Cdk5 activators play nonoverlapping and even opposing roles to govern balanced Cdk5 signaling in the postnatal brain. SIGNIFICANCE STATEMENT: Neuronal network development requires tightly regulated activation of Cyclin-dependent kinase 5 (Cdk5) by two distinct cofactors, p35 and p39. Despite the well-known p35-dependent Cdk5 function, why postnatal neurons express abundant p39 in addition to p35 remained unknown for decades. In this study, we discovered that selective upregulation of p39 is the underlying mechanism that accommodates the increased functional requirement of Cdk5 activation during neuronal differentiation. In addition, we demonstrated that p39 selectively directs Cdk5 to phosphorylate protein substrates essential for axonal development, dendritic spine formation, and synaptogenesis. Moreover, our studies suggest opposing roles of p39 and p35 in synaptic Cdk5 function and epileptic responses, arguing that cooperation between Cdk5 activators maintains balanced Cdk5 signing, which is crucial for postnatal brain function.

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System.

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a ß-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.

Antagonizing effect of CLPABP on the function of HuR as a regulator of ARE-containing leptin mRNA stability and the effect of its depletion on obesity in old male mouse.

Cardiolipin and phosphatidic acid-binding protein (CLPABP) is a pleckstrin homology domain-containing protein and is localized on the surface of mitochondria of cultured cells as a large protein-RNA complex. To analyze the physiological functions of CLPABP, we established and characterized a CLPABP knockout (KO) mouse. Although expression levels of CLPABP transcripts in the developmental organs were high, CLPABP KO mice were normal at birth and grew normally when young. However, old male mice presented a fatty phenotype, similar to that seen in metabolic syndrome, in parallel with elevated male- and age-dependent CLPABP gene expression. One of the reasons for this obesity in CLPABP KO mice is dependence on increases in leptin concentration in plasma. The leptin transcripts were also upregulated in the adipose tissue of KO mice compared with wild-type (WT) mice. To understand the difference in levels of the transcriptional product, we focused on the effect of CLPABP on the stability of mRNA involving an AU-rich element (ARE) in its 3'UTR dependence on the RNA stabilizer, human antigen R (HuR), which is one of the CLPABP-binding proteins. Increase in stability of ARE-containing mRNAs of leptin by HuR was antagonized by the expression of CLPABP in cultured cells. Depletion of CLPABP disturbed the normal subcellular localization of HuR to stress granules, and overexpression of CLPABP induced instability of leptin mRNA by inhibiting HuR function. Consequently, leptin levels in old male mice might be regulated by CLPABP expression, which might lead to body weight control.

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 ß-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-Pd GPI-APs in Arabidopsis (Arabidopsis thaliana). 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-Pd GPI-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-Pd GPI-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-Pd GPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.