An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum

Arabidopsis contains 34 genes that are predicted to encode calcium-dependent protein kinases (CDPKs). CDPK enzymatic activity previously has been detected in many locations in plant cells, including the cytosol, the cytoskeleton, and the membrane fraction. However, little is known about the subcellular locations of individual CDPKs or the mechanisms involved in targeting them to those locations. We investigated the subcellular location of one Arabidopsis CDPK, AtCPK2, in detail. Membrane-associated AtCPK2 did not partition with the plasma membrane in a two-phase system. Sucrose gradient fractionation of microsomes demonstrated that AtCPK2 was associated with the endoplasmic reticulum (ER). AtCPK2 does not contain transmembrane domains or known ER-targeting signals, but does have predicted amino-terminal acylation sites. AtCPK2 was myristoylated in a cell-free extract and myristoylation was prevented by converting the glycine at the proposed site of myristate attachment to alanine (G2A). In plants, the G2A mutation decreased AtCPK2 membrane association by approximately 50%. A recombinant protein, consisting of the first 10 amino acids of AtCPK2 fused to the amino-terminus of beta-glucuronidase, was also targeted to the ER, indicating that the amino terminus of AtCPK2 can specify ER localization of a soluble protein. These results indicate that AtCPK2 is localized to the ER, that myristoylation is likely to be involved in the membrane association of AtCPK2, and that the amino terminal region of AtCPK2 is sufficient for correct membrane targeting.     

The myristoylated amino-terminus of an Arabidopsis calcium-dependent protein kinase mediates plasma membrane localization

Calcium-dependent protein kinases (CDPK) are a major group of calcium-stimulated kinases found in plants and some protists. Many CDPKs are membrane-associated, presumably because of lipid modifications at their amino termini. We investigated the subcellular location and myristoylation of AtCPK5, a member of the Arabidopsis CDPK family. Most AtCPK5 was associated with the plasma membrane as demonstrated by two-phase fractionation of plant microsomes and by in vivo detection of AtCPK5-GFP fusion proteins. AtCPK5 was a substrate for plant N-myristoyltransferase and myristoylation was prevented by converting the glycine at the proposed site of myristate attachment to alanine (G2A). In transgenic plants, a G2A mutation completely abolished AtCPK5 membrane association, indicating that myristoylation was essential for membrane binding. The first sixteen amino acids of AtCPK5 were sufficient to direct plasma membrane localization. In addition, differentially phosphorylated forms of AtCPK5 were detected both in planta and after expression of AtCPK5 in a cell-free plant extract. Our results demonstrate that AtCPK5 is myristoylated at its amino terminus and that myristoylation is required for membrane binding.     

A novel yeast two-hybrid approach to identify CDPK substrates: characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein

Calcium-dependent protein kinases (CDPKs) are sensor-transducer proteins capable of decoding calcium signals in diverse phosphorylation-dependent calcium signaling networks in plants and some protists. Using a novel yeast two-hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11-interacting protein. AtDi19 is a member of a small family of stress-induced genes. The interaction was confirmed using pull-down assays with in vitro translated AtCPK11 and GST-AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca(2+)-dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase-substrate interactions without affecting specificity.     

Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis

Metabolic channeling has been proposed to occur at the entry point into plant phenylpropanoid biosynthesis. To determine whether isoforms of L-Phe ammonia-lyase (PAL), the first enzyme in the pathway, can associate with the next enzyme, the endomembrane-bound cinnamate 4-hydroxylase (C4H), to facilitate channeling, we generated transgenic tobacco (Nicotiana tabacum) plants independently expressing epitope-tagged versions of two PAL isoforms (PAL1 and PAL2) and C4H. Subcellular fractionation and protein gel blot analysis using epitope- and PAL isoform-specific antibodies indicated both microsomal and cytosolic locations of PAL1 but only cytosolic localization of PAL2. However, both PAL isoforms were microsomally localized in plants overexpressing C4H. These results, which suggest that C4H itself may organize the complex for membrane association of PAL, were confirmed using PAL-green fluorescent protein (GFP) fusions with localization by confocal microscopy. Coexpression of unlabeled PAL1 with PAL2-GFP resulted in a shift of fluorescence localization from endomembranes to cytosol in C4H overexpressing plants, whereas coexpression of unlabeled PAL2 with PAL1-GFP did not affect PAL1-GFP localization, indicating that PAL1 has a higher affinity for its membrane localization site than does PAL2. Dual-labeling immunofluorescence and fluorescence energy resonance transfer (FRET) studies confirmed colocalization of PAL and C4H. However, FRET analysis with acceptor photobleaching suggested that the colocalization was not tight.     

A Genome-wide Functional Characterization of Arabidopsis Regulatory Calcium Sensors in Pollen Tubes

Calcium, an ubiquitous second messenger, plays an essential and versatile role in cellular signaling. The diverse function of calcium signals is achieved by an excess of calcium sensors. Plants possess large numbers of calcium sensors, most of which have not been functionally characterized. To identify physiologically relevant calcium sensors in a specific cell type, we conducted a genome-wide functional survey in pollen tubes, for which spatiotemporal calcium signals are well-characterized and required for polarized tip growth. Pollen-specific members of calmodulin (CaM), CaM-like (CML), calcium-dependent protein kinase (CDPK) and calcineurin B-like protein (CBL) families were tagged with green fluorescence protein (GFP) and their localization patterns and overexpression phenotypes were characterized in tobacco pollen tubes. We found that several fusion proteins showed distinct overexpression phenotypes and subcellular localization patterns. CDPK24-GFP was localized to the vegetative nucleus and the generative cell/sperms. CDPK32-GFP caused severe growth depolarization. CBL2-GFP and CBL3-GFP exhibited dynamic patterns of subcellular localization, including several endomembrane compartments, the apical plasma membrane (PM), and cytoskeleton-like structures in pollen tubes. Their overexpression also inhibited pollen tube elongation and induced growth depolarization. These putative calcium sensors are excellent candidates for the calcium sensors responsible for the regulation of calcium homeostasis and calcium-dependent tip growth and growth oscillation in pollen tubes.     

Lack of palmitoylation redirects p59Hck from the plasma membrane to p61Hck-positive lysosomes

Hck, a protein-tyrosine kinase of phagocytes, is the unique member of the Src family expressed under two alternatively translated isoforms differing in their N-terminal site of acylation: p61(Hck) has an additional 21-amino acid sequence comprising a single myristoylation motif, whereas p59(Hck) N terminus has myristoylation and palmitoylation sites. To identify the molecular determinants involved in the targeting of each isoform, they were fused to GFP and expressed in HeLa and CHO cells. p61(Hck) was associated with lysosomal vesicles, whereas p59(Hck) was found at the plasma membrane and to a low extent associated with lysosomes. Their unique N-terminal domains were sufficient to target GFP to the corresponding intracellular compartments. Mutation of the palmitoylation site of p59(Hck) redirected this isoform to lysosomes, indicating that the palmitoylation state governs the association of p59(Hck) with the plasma membrane or with lysosomes. In addition, both isoforms and the nonpalmitoylated p59(Hck) mutant were found on the Golgi apparatus, suggesting a role of this organelle in the subcellular sorting of Hck isoforms. Regarding their subcellular localizations, we propose that bi-acylated p59(Hck) might transduce plasma membrane receptor signals, whereas p61(Hck) and the nonpalmitoylated p59(Hck) might control the biogenesis of phagolysosomes, two functions yet proposed for Hck in phagocytes.     

CDPK-driven changes in the intracellular ROS level and plant secondary metabolism

Heterologous expression of a constitutively active calcium-dependent protein kinase (CDPK) gene was previously shown to increase secondary metabolite production in cultured cells of Rubia cordifolia, but the critical question of how CDPK activates secondary metabolism remains to be answered. In this article, we report that the expression of the Arabidopsis CDPK gene, AtCPK1, in R. cordifolia cells caused moderate and stable elevation of intracellular reactive oxygen species (ROS) levels. In contrast, the non-active, mutated AtCPK1 gene did not cause such an effect. The active AtCPK1 also increased cell size, likely by restricting cell division. These results are consistent with the model in which constitutive expression of AtCPK1 mimics the effects of elicitors, acting on secondary metabolism via the activation of ROS production.     

Characterization of the signal that directs Tom20 to the mitochondrial outer membrane

Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH(2)-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.     

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.     

Cellular localization of dual positional specific maize lipoxygenase-1 in transgenic rice and calcium-mediated membrane association

The dual positional maize lipoxygenase-1 was introduced into rice and T2 transgenic plants were produced. Cellular location of maize lipoxygenase-1 in transgenic rice and effects of calcium ion on membrane association in vitro were analyzed. Localization study by confocal microscopic analysis indicated that the maize lipoxygenase-1 was localized in cytoplasm. Sucrose-density fractionation experiment and in vitro protein transport to chloroplast showed that the maize lipoxygenase-1 can be associated with chloroplast. Secondary structure alignment revealed putative calcium binding sites in the PLAT domain of maize lipoxygenase-1 and the association of the maize lipoxygenase-1 with membranes was mediated by calcium ion in vitro. Our results provide evidences for calcium-mediated translocation of dual positional LOX without chloroplast targeting sequence from cytoplasm to chloroplast in plants for the first time.     

Membrane localization of Arabidopsis acyl-CoA binding protein ACBP2

Cytosolic acyl-CoA binding proteins bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and pool formers. Recently, we have characterized Arabidopsis thaliana cDNAs encoding novel forms of ACBP, designated ACBP1 and ACBP2, that contain a hydrophobic domain at the N-terminus and show conservation at the acyl-CoA binding domain to cytosolic ACBPs. We have previously demonstrated that ACBP1 is membrane-associated in Arabidopsis. Here, western blot analysis of anti-ACBP2 antibodies on A. thaliana protein showed that ACBP2 is located in the microsome-containing membrane fraction and in the subcellular fraction containing large particles (mitochondria, chloroplasts and peroxisomes), resembling the subcellular localization of ACBP1. To further investigate the subcellular localization of ACBP2, we fused ACBP2 translationally in-frame to GFP. By means of particle gene bombardment, ACBP2-GFP and ACBP1-GFP fusion proteins were observed transiently expressed at the plasma membrane and at the endoplasmic reticulum in onion epidermal cells. GFP fusions with deletion derivatives of ACBP1 or ACBP2 lacking the transmembrane domain were impaired in membrane targeting. Our investigations also showed that when the transmembrane domain of ACBP1 or that of ACBP2 was fused with GFP, the fusion protein was targeted to the plasma membrane, thereby establishing their role in membrane targeting. The localization of ACBP1-GFP is consistent with our previous observations using immunoelectron microscopy whereby ACBP1 was localized to the plasma membrane and vesicles. We conclude that ACBP2, like ACBP1, is a membrane protein that likely functions in membrane-associated acyl-CoA transfer/metabolism.     

Identification of proteins that interact with catalytically active calcium-dependent protein kinases from <Emphasis Type="Italic">Arabidopsis</Emphasis>

Calcium-dependent protein kinases (CDPKs) are essential sensor-transducers of calcium signaling pathways in plants. Functional characterization of CDPKs is of great interest because they play important roles during growth, development, and in response to a wide range of environmental stimuli. The Arabidopsis genome encodes 34 CDPKs, but very few substrates of these enzymes have been identified. In this study, we exploited the unique characteristics of CDPKs to develop an efficient approach for the discovery of CDPK-interacting proteins. High-throughput, semi-automated yeast two-hybrid interaction screens with two different cDNA libraries each containing 18 million prey clones were performed using catalytically impaired and constitutively active AtCPK4 and AtCPK11 variants as baits. The use of the constitutively active versions of the CPK baits improved the recovery of positive interacting proteins relative to the wild type kinase. Titration of interaction strength by growth under increasing concentrations of 3-aminotriazole (3-AT), a histidine analog and competitive inhibitor of the His3 gene product, confirmed these results. Possible mechanisms for this observed improvement are discussed. The reproducibility of this approach was assessed by the overlap of several interacting proteins of AtCPK4 and AtCPK11 and the recovery of several putative substrates and indicated that yeast two-hybrid screens using constitutively active and/or catalytically impaired forms of CDPK provides a useful tool to identify potential substrates of the CDPK family and potentially the entire protein kinase superfamily. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo

Certain small outer envelope membrane proteins of chloroplasts are encoded by the nuclear genome without a cleavable N-terminal transit peptide. We investigated in vivo the targeting mechanism of AtOEP7, an Arabidopsis homolog of the small outer envelope membrane protein. AtOEP7 was expressed as a fusion protein with the green fluorescent protein (GFP) either transiently in protoplasts or stably in transgenic plants. In either case, fluorescence microscopy of transformed cells and protein gel blot analysis of fractionated proteins confirmed that the AtOEP7:GFP fusion protein was targeted to the chloroplast outer envelope membrane. In vivo targeting experiments revealed that two regions, the transmembrane domain (TMD) and its C-terminal neighboring seven-amino acid region, were necessary and sufficient for targeting to the chloroplast outer membrane. Substitution of aspartic acid or lysine residues with glycine residues or scrambling of the amino acid sequence of the seven-amino acid region caused mistargeting to the plasma membrane. Although the amino acid sequence of the TMD is not important for targeting, amino acid residues with large side chains inhibited targeting to the chloroplasts and resulted in the formation of large aggregates in the protoplasts. In addition, introduction of a proline residue within the TMD resulted in inhibition of targeting. Finally, a fusion protein, AtOEP7:NLS:GFP, was targeted efficiently to the chloroplast envelope membranes despite the presence of a nuclear localization signal. On the basis of these results, we conclude that the seven-amino acid region and the TMD are determinants for targeting to the chloroplast outer envelope membrane. The seven-amino acid region plays a critical role in AtOEP7 evading the endomembrane system and entering the chloroplast pathway, and the TMD plays critical roles in migration to the chloroplasts and/or subsequent insertion into the membrane.     

Autophosphorylation and subcellular localization dynamics of a salt- and water deficit-induced calcium-dependent protein kinase from ice plant

A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) was isolated from the common ice plant (Mesembryanthemum crystallinum; McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1-70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K0.5 = 0.15 microm). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 6x His-tagged McCPK1.     

Molecular determinants of ciliary membrane localization of Trypanosoma cruzi flagellar calcium-binding protein

The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.     

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.     

Location of divergent region 2 on the three-dimensional structure of cardiac muscle ryanodine receptor/calcium release channel

Ryanodine receptors (RyRs) are a family of calcium release channels found on intracellular calcium-handing organelles. Molecular cloning studies have identified three different RyR isoforms, which are 66-70% identical in amino acid sequence. In mammals, the three isoforms are encoded by three separate genes located on different chromosomes. The major variations among the isoforms occur in three regions, known as divergent regions 1, 2, and 3 (DR1, DR2, and DR3). In the present study, a modified RyR2 (cardiac isoform) cDNA was constructed, into which was inserted a green fluorescent protein (GFP)-encoding cDNA within DR2, specifically after amino acid residue Thr1366 (RyR2(T1366-GFP)). HEK293 cells expressing RyR2(T1366-GFP) cDNAs showed caffeine-sensitive and ryanodine-sensitive calcium release, demonstrating that RyR2(T1366-GFP) forms functional calcium release channels. Cells expressing RyR2(T1366-GFP) were identified readily by the characteristic fluorescence of GFP, indicating that the overall structure of the inserted GFP was retained. Cryo-electron microscopy (cryo-EM) of purified RyR2(T1366-GFP) showed structurally intact receptors, and a three-dimensional reconstruction was obtained by single-particle image processing. The location of the inserted GFP was obtained by comparing this three-dimensional reconstruction to one obtained for wild-type RyR2. The inserted GFP and, consequently Thr1366 within DR2, was mapped on the three-dimensional structure of RyR2 to domain 6, one of the characteristic cytoplasmic domains that form part of the multi-domain "clamp" regions of RyR2. The three-dimensional location of DR2 suggests that it plays roles in the RyR conformational changes that occur during channel gating, and possibly in RyR's interaction with the dihydropyridine receptor in excitation-contraction coupling. This study further demonstrates the feasibility and reliability of the GFP insertion/cryo-EM approach for correlating RyR's amino acid sequence with its three-dimensional structure, thereby enhancing our understanding of the structural basis of RyR function.     

Glycolipid-Enriched Membrane Domains Are Assembled into Membrane Patches by Associating with the Actin Cytoskeleton

Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56(lck) (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.