Arabidopsis Acyl-CoA-Binding Protein ACBP2 Interacts With an Ethylene-Responsive Element-Binding Protein,AtEBP, via its Ankyrin Repeats

Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools. Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats. The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified. Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane. In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A. thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats. This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats. When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane. Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.     

Single amino acid substitutions at the acyl-CoA-binding domain interrupt 14[C]palmitoyl-CoA binding of ACBP2, an Arabidopsis acyl-CoA-binding protein with ankyrin repeats

Cytosolic acyl-CoA-binding proteins (ACBPs) are small proteins (ca. 10 kDa) that bind long-chain acyl-CoAs and are involved in the storage and intracellular transport of acyl-CoAs. Previously, we have characterized an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP, designated ACBP1, demonstrating the existence of a new form of ACBP in plants (M.-L. Chye, Plant Mol. Biol. 38 (1998) 827–838). ACBP1 likely participates in intermembrane lipid transport from the ER to the plasma membrane, where it could maintain a membrane-associated acyl pool (Chye et al., Plant J. 18 (1999) 205–214). Here we report the isolation of cDNAs encoding ACBP2 (M _r 38 479) that shows conservation in the acyl-CoA-binding domain to previously reported ACBPs, and contains ankyrin repeats at its carboxy terminus. These repeats, which likely mediate protein-protein interactions, could constitute a potential docking site in ACBP2 for an enzyme that uses acyl-CoAs as substrate. In vitro binding assays on recombinant (His)₆-ACBP2 expressed in Escherichia coli show that it binds ¹⁴[C]palmitoyl-CoA preferentially to ¹⁴[C]oleoyl-CoA. Analysis of the acyl-CoA-binding domain in ACBP2 was carried out by in vitro mutagenesis. Mutant forms of recombinant (His)₆-ACBP2 with single amino acid substitutions at conserved residues within the acyl-CoA-binding domain were less effective in binding ¹⁴[C]palmitoyl-CoA. Northern blot analysis showed that the 1.6 kb ACBP2 mRNA, like that of ACBP1, is expressed in all plant organs. Analysis of the ACBP2 promoter revealed that, like the ACBP1 promoter, it lacks a TATA box suggesting the possibility of a housekeeping function for ACBP2 in plant lipid metabolism.     

Vasopressin regulated trafficking of a green fluorescent protein-aquaporin 2 chimera in LLC-PK1 cells

 Aquaporin 2 (AQP2) transfected into LLC-PK₁ cells functions as a vasopressin-regulated water channel that recycles between intracellular vesicles and the plasma membrane upon vasopressin stimulation. The green fluorescent protein (GFP) of the jellyfish, Aequorea victoria, was used as an autofluorescent tag to monitor AQP2 trafficking in transfected LLC-PK₁ cells. Two chimeras were constructed, one in which GFP was fused to the amino-terminus of AQP2 [GFP-AQP2(NT)] and the second in which it was fused to the carboxyl-terminus [AQP2-GFP(CT)]. The GFP-AQP2(NT) chimera trafficked in a regulated pathway from intracellular vesicles to the basolateral plasma membrane in response to vasopressin or forskolin stimulation of cells. In contrast, the AQP2-GFP(CT) chimera expressed in LLC-PK₁ cells was localized constitutively on both apical and basolateral plasma membranes. The cellular location of this chimera was not modified by vasopressin or forskolin. Thus, while the GFP-AQP2(NT) chimera will be useful to study AQP2 trafficking in vitro, the abnormal, constitutive membrane localization of the AQP2-GFP(CT) chimera suggests that one or more trafficking signals exist on the carboxyl-terminus of the AQP2 protein. Accepted: 8 April 1998     

Acyl‐CoA‐binding protein 2 binds lysophospholipase 2 and lysoPC to promote tolerance to cadmium‐induced oxidative stress in transgenic Arabidopsis

Lysophospholipids are intermediates of phospholipid metabolism resulting from stress and lysophospholipases detoxify lysophosphatidylcholine (lysoPC). Many lysophospholipases have been characterized in mammals and bacteria, but few have been reported from plants. Arabidopsis thaliana lysophospholipase 2 (lysoPL2) (At1g52760) was identified as a protein interactor of acyl‐CoA‐binding protein 2 (ACBP2) in yeast two‐hybrid analysis and co‐immunoprecipitation assays. BLASTP analysis indicated that lysoPL2 showed ～35% amino acid identity to the lysoPL1 family. Co‐localization of autofluorescence‐tagged lysoPL2 and ACBP2 by confocal microscopy in agroinfiltrated tobacco suggests the plasma membrane as a site for their subcellular interaction. LysoPL2 mRNA was induced by zinc (Zn) and hydrogen peroxide (H₂O₂), and lysoPL2 knockout mutants showed enhanced sensitivity to Zn and H₂O₂ in comparison to wild type. LysoPL2‐overexpressing Arabidopsis was more tolerant to H₂O₂ and cadmium (Cd) than wild type, suggesting involvement of lysoPL2 in phospholipid repair following lipid peroxidation arising from metal‐induced stress. Lipid hydroperoxide (LOOH) contents in ACBP2‐overexpressors and lysoPL2‐overexpressors after Cd‐treatment were lower than wild type, indicating that ACBP2 and lysoPL2 confer protection during oxidative stress. A role for lysoPL2 in lysoPC detoxification was demonstrated when recombinant lysoPL2 was observed to degrade lysoPC in vitro. Filter‐binding assays and Lipidex competition assays showed that (His)₆‐ACBP2 binds lysoPC in vitro. Binding was disrupted in a (His)₆‐ACBP2 derivative lacking the acyl‐CoA‐binding domain, confirming that this domain confers lysoPC binding. These results suggest that ACBP2 can bind both lysoPC and lysoPL2 to promote the degradation of lysoPC in response to Cd‐induced oxidative stress.     

Functional cooperation of two independent targeting domains in syntaxin 6 is required for its efficient localization in the trans-golgi network of 3T3L1 adipocytes

To identify the targeting domains of syntaxin 6 responsible for its localization to the trans-Golgi network (TGN), we examined the subcellular distribution of enhanced green fluorescent protein (EGFP) epitope-tagged syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncation/deletion mutants in 3T3L1 adipocytes. Expression of EGFP-syntaxin 6 resulted in a perinuclear distribution identical to endogenous syntaxin 6 as determined both by confocal fluorescence microscopy and subcellular fractionation. Furthermore, both the endogenous and the expressed EGFP-syntaxin 6 fusion protein were localized to a brefeldin A-insensitive but okadaic acid-sensitive compartment characteristic of the TGN. In contrast, EGFP-syntaxin 6 constructs lacking the H2 domain were excluded from the TGN and were instead primarily localized to the plasma membrane. Although syntaxin 4 was localized to the plasma membrane, syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncations containing the H2 domain of syntaxin 6 were predominantly directed to the TGN. Importantly, the syntaxin 6 H2 domain fused to the transmembrane domain of syntaxin 4 was also localized to the TGN, demonstrating that the H2 domain was sufficient to confer TGN localization. In addition to the H2 domain, a tyrosine-based plasma membrane internalization signal (YGRL) was identified between the H1 and H2 domains of syntaxin 6. Deletion of this sequence resulted in the accumulation of the EGFP-syntaxin 6 reporter construct at the plasma membrane. Together, these data demonstrate that syntaxin 6 utilizes two distinct domains to drive its specific subcellular localization to the TGN.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.     

Molecular characterization of soybean ribosomal protein S13 targeted to the nucleus

We isolated the full-length genomic clone of STLI25 encoding RPS13 (ribosomal protein S13) in soybean. Genomic DNA structure of SLTI25 is similar to that of Arabidopsis ribosomal protein S13. RNA expression of SLTI25 was induced by salt, ABA, or wounding stress, but reduced by dehydration stress. To determine the subcellular localization of the gene product fused to GFP, we were able to confirm that SLTI25-GFP was restricted to the nucleus. By domain swapping analysis, it was shown that C-terminal region of SLTI25 with a putative nucleus localization signal was necessary and sufficient for nucleus targeting of the fusion protein in plants. This new findings provide an evidence that SLTI25 is targeted to the nucleus for the ribosome subunit assembly in plants. Published in Russian in Fiziologiya Rastenii, 2009, vol. 56, No. 3, pp. 445–452 This text was submitted by the authors in English. These authors contributed equally to the work.     

Isolation of a gene encoding Arabidopsis membrane-associated acyl-CoA binding protein and immunolocalization of its gene product

Until recently, only cytosolic acyl-CoA binding proteins (ACBPs) have been characterized. The isolation of an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP that accumulates in developing seeds, designated ACBP1, has provided evidence for the existence of membrane-associated forms of ACBPs (Chye, 1998, Plant Mol. Biol. 38, 827-838). We now report on the isolation of its corresponding gene from an A. thaliana Columbia genomic library using the ACBP1 cDNA as a hybridization probe. Nucleotide sequence analysis of Arabidopsis ACBP1 showed that its promoter lacks a TATA box, resembling the promoters of rat, Drosophila and human genes encoding cytosolic ACBP and suggesting that it is a housekeeping gene. We show by Western blot analysis that ACBP1 expression in developing seeds coincides with lipid deposition and that homologues of membrane-associated ACBP1 exist in other plants. Using light microscopy, we show that ACBP1 is strongly expressed in the embryo at the cotyledons, hypocotyl, procambium of the axis and in most peripheral cells of the cotyledons and hypocotyl. Immunogold labelling localized ACBP1 to vesicles, to the plasma membrane especially at epidermal cells of heart, torpedo and cotyledonary stage embryos, and to the cell wall of the outer integument cells at the seed coat. Our results suggest that ACBP1 is involved in intermembrane lipid transport from the ER via vesicles to the plasma membrane where it could maintain a membrane-associated acyl pool; its immunolocalization to the cell wall of outer integument cells at the seed coat suggests a role in cuticle and cutin formation.     

Overexpression of Arabidopsis Acyl-CoA Binding Protein ACBP3 Promotes Starvation-Induced and Age-Dependent Leaf Senescence

In Arabidopsis thaliana, a family of six genes (ACBP1 to ACBP6) encodes acyl-CoA binding proteins (ACBPs). Investigations on ACBP3 reported here show its upregulation upon dark treatment and in senescing rosettes. Transgenic Arabidopsis overexpressing ACBP3 (ACBP3-OEs) displayed accelerated leaf senescence, whereas an acbp3 T-DNA insertional mutant and ACBP3 RNA interference transgenic Arabidopsis lines were delayed in dark-induced leaf senescence. Acyl-CoA and lipid profiling revealed that the overexpression of ACBP3 led to an increase in acyl-CoA and phosphatidylethanolamine (PE) levels, whereas ACBP3 downregulation reduced PE content. Moreover, significant losses in phosphatidylcholine (PC) and phosphatidylinositol, and gains in phosphatidic acid (PA), lysophospholipids, and oxylipin-containing galactolipids (arabidopsides) were evident in 3-week-old dark-treated and 6-week-old premature senescing ACBP3-OEs. Such accumulation of PA and arabidopsides (A, B, D, E, and G) resulting from lipid peroxidation in ACBP3-OEs likely promoted leaf senescence. The N-terminal signal sequence/transmembrane domain in ACBP3 was shown to be essential in ACBP3-green fluorescent protein targeting and in promoting senescence. Observations that recombinant ACBP3 binds PC, PE, and unsaturated acyl-CoAs in vitro and that ACBP3 overexpression enhances degradation of the autophagy (ATG)-related protein ATG8 and disrupts autophagosome formation suggest a role for ACBP3 as a phospholipid binding protein involved in the regulation of leaf senescence by modulating membrane phospholipid metabolism and ATG8 stability in Arabidopsis. Accelerated senescence in ACBP3-OEs is dependent on salicylic acid but not jasmonic acid signaling.     

Sorting of GFP Tagged NtSyr1, an ABA Related Syntaxin

Exocytosis molecular mechanisms in plant cells are not fully understood. The full characterization of molecular determinants, such as SNAREs, for the specificity in vesicles delivery to the plasma membrane should shed some light on these mechanisms. Nicotiana tabacum Syntaxin 1 (NtSyr1 or SYP121) is a SNARE protein required for ABA control of ion channels and appears involved in the exocytosis of exogenous markers.NtSyr1 is mainly localized on the plasma membrane, but when over expressed the protein also appears on endomembranes. Since NtSyr1 is a tail-anchored protein inserted into the target membrane post-translationally, it is not clear whether its initial anchoring site is the ER or the plasma membrane.In this study, we investigated the sorting events of NtSyr1 in vivo using its full-length cDNA or its C-terminal domain, fused to a GFP tag and transiently expressed in protoplasts or in the leaves of Nicotiana tabacum cv. SR1. Five chimeras were produced of which two were useful to investigate the protein sorting within the endomembrane system. One (GFP-H3M) had a dominant negative effect on exocytosis; the other one (SP1-GFP) resulted in a slow targeting to the same localization of the full-length chimera (GFP-SP1). The insertion of signal peptides on SP1-GFP further characterized the insertion site for this protein. Our data indicates that NtSyr1 is firstly anchored on ER membrane and then sorted to plasma membrane.Key Words: syntaxins, SNAREs, GFP tagging, exocytosis, secretion, protoplasts, dominant negative mutant     

Light-regulated Arabidopsis ACBP4 and ACBP5 encode cytosolic acyl-CoA-binding proteins that bind phosphatidylcholine and oleoyl-CoA ester

In Arabidopsis thaliana, six genes encode acyl-CoA-binding proteins (ACBPs) that show conservation of an acyl-CoA-binding domain. These ACBPs display varying affinities for acyl-CoA esters, suggesting of different cellular roles. We have recently reported that three members (ACBP4, ACBP5 and ACBP6) are subcellularly localized to the cytosol by biochemical fractionation, confocal microscopy of transgenic Arabidopsis expressing autofluorescence-tagged fusions and immuno-electron microscopy using ACBP-specific antibodies. In this study, we observed by Northern blot analysis that ACBP4 and ACBP5 mRNAs in rosettes were up-regulated by light and dampened-off in darkness, mimicking FAD7 which encodes omega-3-fatty acid desaturase, an enzyme involved in plastidial lipid metabolism. Results from in vitro binding assays indicate that recombinant ACBP4 and ACBP5 proteins bind [¹⁴C]oleoyl-CoA esters better than recombinant ACBP6, suggesting that light-regulated ACBP4 and ACBP5 encode cytosolic ACBPs that are potential candidates for the intracellular transport of oleoyl-CoA ester exported from the chloroplast to the endoplasmic reticulum for the biosynthesis of non-plastidial membrane lipids. Nonetheless, His-tagged ACBP4 and ACBP5 resemble ACBP6 in their ability to bind phosphatidylcholine suggesting that all three ACBPs are available for the intracellular transfer of phosphatidylcholine.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal     

Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.     

Retention and mobility of the mammalian lamin B receptor in the plant nuclear envelope

Background information. In a previous study, we showed that GFP (green fluorescent protein) fused to the N‐terminal 238 amino acids of the mammalian LBR (lamin B receptor) localized to the NE (nuclear envelope) when expressed in the plant Nicotiana tabacum. The protein was located in the NE during interphase and migrated with nuclear membranes during cell division. Targeting and retention of inner NE proteins requires several mechanisms: signals that direct movement through the nuclear pore complex, presence of a transmembrane domain or domains and retention by interaction with nuclear or nuclear‐membrane constituents. Results. Binding mutants of LBR—GFP were produced to investigate the mechanisms for the retention of LBR in the NE. FRAP (fluorescence recovery after photobleaching) analysis of mutant and wild‐type constructs was employed to examine the retention of LBR—GFP in the plant NE. wtLBR—GFP (wild‐type LBR—GFP) was shown to have significantly lower mobility in the NE than the lamin‐binding domain deletion mutant, which showed increased mobility in the NE and was also localized to the endoplasmic reticulum and punctate structures in some cells. Modification of the chromatin‐binding domain resulted in the localization of the protein in nuclear inclusions, in which it was immobile. Conclusions. As expression of truncated LBR—GFP in plant cells results in altered targeting and retention compared with wtLBR—GFP, we conclude that plant cells can recognize the INE (inner NE)‐targeting motif of LBR. The altered mobility of the truncated protein suggests that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.     

The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo

Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules.     

Insulin-regulated trafficking of dual-labeled glucose transporter 4 in primary rat adipose cells

In isolated rat adipose cells, physiologically relevant insulin target cells, glucose transporter 4 (GLUT4) subcellular trafficking can be assessed by transfection of exofacially HA-tagged GLUT4. To simultaneously visualize the transfected GLUT4, we fused GFP with HA-GLUT4. With the resulting chimeras, GFP-HA-GLUT4 and HA-GLUT4-GFP, we were able to visualize for the first time the cell-surface localization, total expression, and intracellular distribution of GLUT4 in a single cell. Confocal microscopy reveals that the intracellular proportions of both GFP-HA-GLUT4 and HA-GLUT4-GFP are properly targeted to the insulin-responsive aminopeptidase-positive vesicles. Dynamic studies demonstrate close similarities in the trafficking kinetics between the two constructs and with native GLUT4. However, while the basal subcellular distribution of HA-GLUT4-GFP and the response to insulin are indistinguishable from those of HA-GLUT4 and endogenous GLUT4, most of the GFP-HA-GLUT4 is targeted to the plasma membrane with little further insulin response. Thus, HA-GLUT4-GFP will be useful to study GLUT4 trafficking in vivo while GFP on the N-terminus interferes with intracellular retention.     

Hydrophobic regions adjacent to transmembrane domains 1 and 5 are important for the targeting of the 70-kDa peroxisomal membrane protein

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH(2)-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH(2)-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH(2)-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile(307)/Leu(308)) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.     

Arabidopsis cDNA encoding a membrane-associated protein with an acyl-CoA binding domain

Acyl-CoA binding proteins (ACBPs) are small (ca. 10 kDa) highly-conserved cytosolic proteins that bind long-chain acyl-CoAs. A novel cDNA encoding ACBP1, a predicted membrane protein of 24.1 kDa with an acyl-CoA binding protein domain at its carboxy terminus, was cloned from Arabidopsis thaliana. At this domain, ACBP1 showed 47% amino acid identity to Brassica ACBP and 35% to 40% amino acid identity to yeast, Drosophila, bovine and human ACBPs. Recombinant (His)6-ACBP1 fusion protein was expressed in Escherichia coli and was shown to bind 14[C]oleoyl-CoA. A hydrophobic domain, absent in the 10 kDa ACBPs, was located at the amino terminus of ACBP1. Using antipeptide polyclonal antibodies in western blot analysis, ACBP1 was shown to be a membrane-associated glycosylated protein with an apparent molecular mass of 33 kDa. The ACBP1 protein was also shown to accumulate predominantly in siliques and was localized to the seed within the silique. These results suggest that the biological role of ACBP1 is related to lipid metabolism in the seed, presumably in which acyl-CoA esters are involved. Northern blot analysis showed that the 1.4 kb ACBP1 mRNA was expressed in silique, root, stem, leaf and flower. Results from Southern blot analysis of genomic DNA suggest the presence of at least two genes encoding ACBPs in Arabidopsis.