Identification and functional characterization of the BAG protein family in Arabidopsis thaliana

The genes that control mammalian programmed cell death are conserved across wide evolutionary distances. Although plant cells can undergo apoptosis-like cell death, plant homologs of mammalian regulators of apoptosis have, in general, not been found. This is in part due to the lack of primary sequence conservation between animal and putative plant regulators of apoptosis. Thus, alternative approaches beyond sequence similarities are required to find functional plant homologs of apoptosis regulators. Here, we present the results of using advanced bioinformatic tools to uncover the Arabidopsis family of BAG proteins. The mammalian BAG (Bcl-2-associated athanogene) proteins are a family of chaperone regulators that modulate a number of diverse processes ranging from proliferation to growth arrest and cell death. Such proteins are distinguished by a conserved BAG domain that directly interacts with Hsp70 and Hsc70 proteins to regulate their activity. Our searches of the Arabidopsis thaliana genome sequence revealed seven homologs of the BAG protein family. We further show that plant BAG family members are also multifunctional and remarkably similar to their animal counterparts, as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development.     

Identification and functional characterization of ankyrin-repeat family protein ANKRA as a protein interacting with BKCa channel

Ankyrin-repeat family A protein (ANKRA) was originally cloned in mouse as an interacting protein to megalin, a member of low-density lipoprotein receptor superfamily. Here, we report that the isolation of rat ANKRA as a new binding partner for the alpha-subunit of rat large-conductance Ca2+-activated K+ channel (rSlo). We mapped the binding region of each protein by using yeast two-hybrid and in vitro binding assays. ANKRA expressed together with rSlo channels were colocalized near the plasma membrane and coimmunoprecipitated in transfected cells. We also showed that BKCa channel in rat cerebral cortex coprecipitated with rANKRA and colocalized in cultured rat hippocampal neuron. Although the coexpression of ANKRA did not affect the surface expression of rSlo, the gating kinetics of rSlo channel was significantly altered and the effects were highly dependent on the intracellular calcium. These results indicate that ANKRA could modulate the excitability of neurons by binding directly to endogenous BKCa channel and altering its gating kinetics in a calcium-dependent manner.     

Identification of proteins interacting with the Arabidopsis Cdc2aAt protein

Cyclin-dependent kinases (CDK5) control the progression throughthe cell cycle. Using a two-hybrid approach, two clones encodingproteins interacting with the Arabidopsis thailana CDK Cdc2aAtwere identified. One clone encoded a novel putative substrateof Cdc2aAt, whereas the second clone was identified as a D-typecyclin (cycDl;1). Key words: Arabidopsis thaliana, cell cycle, cyclin, cyclindependent kinases, yeast two-hybrid screening     

Identification and functional analysis of the MOC1 interacting protein 1

Rice tillering is one of the most important agronomic traits that determine grain yields.Our previous study has demonstrated that the MONOCULM1(MOC1)gene is a key component that controls the formation of rice tiller buds.To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering.we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins(MIPs).Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo.The overexpression of MIP1 resulted in enhanced tillering and reduced plant height.In-depth characterization of the context of MIP1 and MOC1 would further our understanding of molecular regulatory mechanisms of rice tillering.     

Identification and functional analysis of the MOC1 interacting protein 1

Rice tillering is one of the most important agronomic traits that determine grain yields.Our previous study has demonstrated that the MONOCULM1 (MOC1) gene is a key component that controls the formation of rice tiller buds.To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering,we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins (MIPs).Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo.The overexpression of MIP1 res...     

The PEROXIN11 protein family controls peroxisome proliferation in Arabidopsis

PEROXIN11 (PEX11) is a peroxisomal membrane protein in fungi and mammals and was proposed to play a major role in peroxisome proliferation. To begin understanding how peroxisomes proliferate in plants and how changes in peroxisome abundance affect plant development, we characterized the extended Arabidopsis thaliana PEX11 protein family, consisting of the three phylogenetically distinct subfamilies PEX11a, PEX11b, and PEX11c to PEX11e. All five Arabidopsis PEX11 proteins target to peroxisomes, as demonstrated for endogenous and cyan fluorescent protein fusion proteins by fluorescence microscopy and immunobiochemical analysis using highly purified leaf peroxisomes. PEX11a and PEX11c to PEX11e behave as integral proteins of the peroxisome membrane. Overexpression of At PEX11 genes in Arabidopsis induced peroxisome proliferation, whereas reduction in gene expression decreased peroxisome abundance. PEX11c and PEX11e, but not PEX11a, PEX11b, and PEX11d, complemented to significant degrees the growth phenotype of the Saccharomyces cerevisiae pex11 null mutant on oleic acid. Heterologous expression of PEX11e in the yeast mutant increased the number and reduced the size of the peroxisomes. We conclude that all five Arabidopsis PEX11 proteins promote peroxisome proliferation and that individual family members play specific roles in distinct peroxisomal subtypes and environmental conditions and possibly in different steps of peroxisome proliferation.     

Identification of a novel small Arabidopsis protein interacting with gamma-tubulin complex protein 3

In higher plants, microtubules (MTs) show dynamic structural changes during cell cycle and development progression. A precise control of MT nucleation at dispersed sites is one way used to regulate the cytoskeletal organization. Some gamma-tubulin complex proteins (GCPs) were previously identified in Arabidopsis thaliana (At). They are directly involved in the nucleation process. Nevertheless, no additional player which may anchor the nucleating complex or regulate the nucleation activity has been found in plant cells so far. Therefore, our aim was the identification of Arabidopsis proteins interacting with MT nucleating complexes and particularly with AtGCP3. Performing a yeast two-hybrid screen, we discovered a new protein which we called AtGCP3 Interacting Protein 1 (AtGIP1). The possible role of this protein during the nucleation process is discussed.     

Identification and characterization of a UDP-D-glucuronate 4-epimerase in Arabidopsis

One of the major sugars present in the plant cell wall is d-galacturonate, the dominant monosaccharide in pectic polysaccharides. Previous work indicated that one of the activated precursors necessary for the synthesis of pectins is UDP-d-galacturonate, which is synthesized from UDP-d-glucuronate by a UDP-d-glucuronate 4-epimerase (GAE). Here, we report the identification, cloning and characterization of a GAE6 from Arabidopsis thaliana. Functional analysis revealed that this enzyme converts UDP-d-glucuronate to UDP-d-galacturonate in vitro. An expression analysis of this epimerase and its five homologs in the Arabidopsis genome by quantitative RT-PCR and promoter::GUS fusions indicated differential expression of the family members in plant tissues and expression of all isoforms in the developing pollen of A. thaliana.     

Identification of proteins interacting with Arabidopsis ACD11

The Arabidopsis ACD11 gene encodes a sphingosine transfer protein and was identified by the accelerated cell death phenotype of the loss of function acd11 mutant, which exhibits heightened expression of genes involved in the disease resistance hypersensitive response (HR). We used ACD11 as bait in a yeast two-hybrid screen of an Arabidopsis cDNA library to identify ACD11 interacting proteins. One interactor identified is a protein of unknown function with an RNA recognition motif (RRM) designated BPA1 (binding partner of ACD11). Co-immunoprecipitation experiments confirmed the ACD11-BPA1 interactions in vivo and in vitro. Two other ACD11 interactors (PRA7 and PRA8) are homologous to each other and to mammalian PRA1, and both were subsequently shown to interact with BPA1 in yeast. A fourth interactor (VAP27-1) is homologous to mammalian VAP-A, and was found to interact more strongly with a homolog of ACD11 than ACD11 itself. All interactors were shown to be associated with membrane fractions, suggesting that ACD11 function could be related to the regulation of membrane compartments.     

Identification and characterization of the ICP22 protein of equine herpesvirus 1.

The equine herpesvirus 1 (EHV-1) homolog of herpes simplex virus type 1 ICP22 is differently expressed from the fourth open reading frame of the inverted repeat (IR4) as a 1.4-kb early mRNA and a 1.7-kb late mRNA which are 3' coterminal (V. R. Holden, R. R. Yalamanchili, R. N. Harty, and D. J. O'Callaghan, J. Virol. 66:664-673, 1992). To extend the characterization of IR4 at the protein level, the synthesis and intracellular localization of the IR4 protein were investigated. Antiserum raised against either a synthetic peptide corresponding to amino acids 270 to 286 or against a TrpE-IR4 fusion protein (IR4 residues 13 to 150) was used to identify the IR4 protein. Western immunoblot analysis revealed that IR4 is expressed abundantly from an open reading frame composed of 293 codons as a family of proteins that migrate between 42 to 47 kDa. The intracellular localization of IR4 was examined by cell fractionation, indirect immunofluorescence, and laser-scanning confocal microscopy. These studies revealed that IR4 is localized predominantly in the nucleus and is dispersed uniformly throughout the nucleus. Interestingly, when IR4 is expressed transiently in COS-1 or LTK- cells, a punctate staining pattern within the nucleus is observed by indirect immunofluorescence. Cells transfected with an IR4 mutant construct that encodes a C-terminal truncated (19 amino acids) IR4 protein exhibited greatly reduced intranuclear accumulation of the IR4 protein, indicating that this domain possesses an important intranuclear localization signal. Western blot analysis of EHV-1 virion proteins revealed that IR4 proteins are structural components of the virions. Surprisingly, the 42-kDa species, which is the least abundant and the least modified form of the IR4 protein family in infected cell extracts, was the most abundant IR4 protein present in purified virions.     

Identification and functional characterization of a novel barnacle cement protein

Barnacle attachment to various foreign materials in water is guided by an extracellular multiprotein complex. A 19 kDa cement protein was purified from the Megabalanus rosa cement, and its cDNA was cloned and sequenced. The gene was expressed only in the basal portion of the animal, where the histologically identified cement gland is located. The sequence of the protein showed no homology to other known proteins in the databases, indicating that it is a novel protein. Agreement between the molecular mass determined by MS and the molecular weight estimated from the cDNA indicated that the protein bears no post-translational modifications. The bacterial recombinant was prepared in soluble form under physiologic conditions, and was demonstrated to have underwater irreversible adsorption activity to a variety of surface materials, including positively charged, negatively charged and hydrophobic ones. Thus, the function of the protein was suggested to be coupling to foreign material surfaces during underwater attachment. Homologous genes were isolated from Balanus albicostatus and B. improvisus, and their amino acid compositions showed strong resemblance to that of M. rosa, with six amino acids, Ser, Thr, Ala, Gly, Val and Lys, comprising 66-70% of the total, suggesting that such a biased amino acid composition may be important for the function of this protein.     

Identification and characterization of the domain structure of bacteriophage P22 coat protein.

The bacteriophage P22 serves as a model for assembly of icosahedral dsDNA viruses. The P22 procapsid, which constitutes the precursor for DNA packaging, is built from 420 copies of a single coat protein with the aid of stoichiometric amounts of scaffolding protein. Upon DNA entry, the procapsid shell expands and matures into a stable virion. It was proposed that expansion is mediated by hinge bending and domain movement. We have used limited proteolysis to map the dynamic stability of the coat protein domain structures. The coat protein monomer is susceptible to proteolytic digestion, but limited proteolysis by small quantities of elastase or chymotrypsin yielded two metastable fragments (domains). The N-terminal domain (residues 1-180) is linked to the C-terminal domain (residues 205-429) by a protease-susceptible loop (residues 180-205). The two domains remain associated after the loop cleavage. Although only a small change of secondary structure results from the loop cleavage, both tertiary interdomain contacts and subunit thermostability are diminished. The intact loop is also required for assembly of the monomeric coat protein into procapsids. Upon assembly, coat protein becomes largely protease-resistant, baring cleavage within the loop region of about half of the subunits. Loop cleavage decreases the stability of the procapsids and facilitates heat-induced shell expansion. Upon expansion, the loop becomes protease-resistant. Our data suggest the loop region becomes more ordered during assembly and maturation and thereby plays an important role in both of these stages.     

Identification and functional analysis of human Tom22 for protein import into mitochondria

Mitochondria have a receptor complex in the outer membrane which recognizes and translocates mitochondrial proteins synthesized in the cytosol. We report here the identification and functional analysis of human Tom22 (hTom22). hTom22 has an N-terminal negatively charged region exposed to the cytosol, a putative transmembrane region, and a C-terminal intermembrane space region with little negative charge. Tom22 forms a complex with Tom20, and its cytosolic domain functions as an import receptor as in fungi. An import inhibition assay, using pre-ornithine transcarbamylase (pOTC) derivatives and a series of hTom22 deletion mutants, showed that the C-terminal segment of the cytosolic domain is important for presequence binding, whereas the N-terminal domain is important for binding to the mature portion of pOTC. No evidence for pOTC interaction with the Tom22 intermembrane space domain was obtained. Binding studies revealed that the presequence is critical for pOTC binding to Tom20, whereas both the presequence and mature portion are important for binding to Tom22. A cell-free immunoprecipitation assay indicated that an internal segment of the Tom22 cytosolic domain is important for interaction with Tom20.     

Arabidopsis 22-kilodalton peroxisomal membrane protein. Nucleotide sequence analysis and biochemical characterization.

We sequenced and characterized PMP22 (22-kD peroxisomal membrane protein) from Arabidopsis, which shares 28% to 30% amino acid identity and 55% to 57% similarity to two related mammalian peroxisomal membrane proteins, PMP22 and Mpv17. Subcellular fractionation studies confirmed that the Arabidopsis PMP22 is a genuine peroxisomal membrane protein. Biochemical analyses established that the Arabidopsis PMP22 is an integral membrane protein that is completely embedded in the lipid bilayer. In vitro import assays demonstrated that the protein is inserted into the membrane posttranslationally in the absence of ATP, but that ATP stimulates the assembly into the native state. Arabidopsis PMP22 is expressed in all organs of the mature plant and in tissue-cultured cells. Expression of PMP22 is not associated with a specific peroxisome type, as it is detected in seeds and throughout postgerminative growth as cotyledon peroxisomes undergo conversion from glyoxysomes to leaf-type peroxisomes. Although PMP22 shows increased accumulation during the growth of young seedlings, its expression is not stimulated by light.     

Identification and characterization of two functional domains of the hemolysin translocator protein HlyD

Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC. Two regions of HlyD were studied. The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure. This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species. Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function. The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated. Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus. The second region of HIyD (region B), located between amino acids Leul27 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC. Deletion of this region abolishes secretion of hemolysin. This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HIyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus.     

Cloning and functional characterization of a formin-like protein (AtFH8) from Arabidopsis

The actin cytoskeleton is required for many cellular processes in plant cells. The nucleation process is the rate-limiting step for actin assembly. Formins belong to a new class of conserved actin nucleator, which includes at least 2 formin homology domains, FH1 and FH2, which direct the assembly of unbranched actin filaments. The function of plant formins is quite poorly understood. Here, we provide the first biochemical study of the function of conserved domains of a formin-like protein (AtFH8) from Arabidopsis (Arabidopsis thaliana). The purified recombinant AtFH8(FH1FH2) domain has the ability to nucleate actin filaments in vitro at the barbed end and caps the barbed end of actin filaments, decreasing the rate of subunit addition and dissociation. In addition, purified AtFH8(FH1FH2) binds actin filaments and severs them into short fragments. The proline-rich domain (FH1) of the AtFH8 binds directly to profilin and is necessary for nucleation when actin monomers are profilin bound. However, profilin inhibits the nucleation mediated by AtFH8(FH1FH2) to some extent, but increases the rate of actin filament elongation in the presence of AtFH8(FH1FH2). Moreover, overexpression of the full-length AtFH8 in Arabidopsis causes a prominent change in root hair cell development and its actin organization, indicating the involvement of AtFH8 in polarized cell growth through the actin cytoskeleton.