The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities.

BACKGROUND: HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and -2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants. RESULTS: Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and -2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage. CONCLUSIONS: The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways.

G-protein-coupled receptor-associated A-kinase anchoring proteins AKAP5 and AKAP12: differential trafficking and distribution.

A-kinase Anchoring Proteins (AKAPs) define an expanding group of scaffold proteins that display a signature binding site for the RI/RII subunit of protein kinase A. AKAP5 and AKAP12 are multivalent (with respect to protein kinases and phosphatases) and display the ability to associate with the prototypic member of G protein-coupled receptors, the beta(2)-adrenergic receptor. We probed the relative abundance, subcellular distribution and localization of AKAP5 and AKAP12 in human embryonic kidney HEK293 and epidermoid carcinoma A431 cells. HEK293 cells are relatively rich in AKAP5 (found mostly in association with the cell membrane); whereas A431 cells are rich in AKAP12 (found distributed both in the cytoplasm and in association with the cell membrane). In biochemical analysis of subcellular fractions and in whole-cell imaging, the membrane localization of AKAP5 was decreased in response to treating cells with the beta-adrenergic agonist isoproterenol, whereas membrane association of AKAP12 was increased initially in response to agonist treatment. These data demonstrate quantitatively a clearly different pattern of AKAP-receptor association for AKAP5 versus AKAP12. AKAP5 remains associated with its G-protein-coupled receptor, at the cell membrane, docked with the receptor during agonist-induced internalization and later receptor recycling after agonist wash-out. AKAP12-receptor docking, in contrast, is dynamic, driven by agonist stimulation (accounting for movement of AKAP12 from the cytoplasm to the cell membrane). AKAP12 then is internalized with the beta(2)-adrenergic receptor, but segregates away from the G-protein-coupled receptor upon recycling of the internalized receptor to the cell membrane. Thus these homologous, AKAPs that dock G-protein-coupled receptors have markedly different patterns of trafficking, docking, and re-distribution.

Nucleocytoplasmic-localized acyltransferases catalyze the malonylation of 7-O-glycosidic (iso)flavones in Medicago truncatula.

(Iso)flavonoids are commonly accumulated as malonylated or acetylated glycoconjugates in legumes. Sequence analysis on EST database of the model legume Medicago truncatula enabled us to identify nine cDNA sequences encoding BAHD super-family enzymes that are distinct from the most of the characterized anthocyanin/flavonol acyltransferase genes in other species. Functional characterization revealed that three of these corresponding enzymes, MtMaT1, 2 and 3, specifically recognize malonyl CoA as an acyl donor and catalyze the malonylation of a range of isoflavone 7-O-glucosides in vitro. These malonyltransferase genes displayed distinct tissue-specific expression patterns and responded differentially to biotic and abiotic stresses. Consistent with gene expression, the level of the accumulated malonyl isoflavone glucoside was altered in the roots of M. truncatula grown under normal and drought-stressed conditions. Overexpression of the MtMaT1 gene in a previously engineered Arabidopsis line that accumulates genistein glycosides (Proc. Natl Acad. Sci. USA, 99, 2002:14578) led to a malonylated product. Confocal microscopy of the transiently expressed MtMaT1-GFP fusion revealed strong fluorescence in both the cytoplasm and nucleus of M. truncatula and tobacco leaf cells. A truncated MtMaT1 lacking the C-terminal polypeptide of 110 amino acid residues that include the DFGWG motif, the single conserved sequence signature of BAHD super-family members, retained considerable catalytic efficiency, but showed an altered optimum pH preference for maximum activity. Such C-terminal polypeptide deletion or deletion of the DFGWG motif alone led to improper folding of the transiently expressed GFP fusion protein in living cells, and impaired nuclear localization of the enzyme.

Tissue engineering bone formation in novel recombinant human bone morphogenic protein 2-atelocollagen composite scaffolds.

BACKGROUND: Bone morphogenic proteins (BMPs) are important bone-induction factors, and the development of a suitable carrier for BMPs is a critical step to achieve osteoinductive function. The aims of the present study were to evaluate, at the cellular and molecular levels, the feasibility of recombinant human BMP-2 (rhBMP-2)-collagen composite scaffold and its efficiency for carrying BMP-2 in ectopic bone formation in rats. METHODS: Scaffolds with (test) or without rhBMP-2 (control) were made and implanted into the calf muscle of 16 5-week-old rats. The tissue responses to the scaffolds were examined by histology. Masson's trichrome and von Kossa stainings were performed to examine collagen matrix deposition and calcification at 3, 7, 10, and 14 days. Expressions of bone phenotypic markers, alkaline phosphatase, osteocalcin, osteopontin, and bone sialoprotein were detected by reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: No detectable adverse responses were noted around the implanted scaffolds, and the area of the resorbed scaffold had been replaced by young connective tissue by 3 to 7 days in both groups. In the rhBMP-2 composite scaffold, collagen matrix deposition was found in the implanted site on day 7 and initial signs of endochondral differentiation also appeared. Mineralization and the expressions of key bone proteins were demonstrated in chondroblasts and osteoblasts at 7 to 14 days. Molecular cascades of bone induction were not shown in control specimens. CONCLUSION: The rhBMP-2-atelocollagen scaffold showed excellent biocompatibility and possessed a bone-inducing capacity in rat within 2 weeks, and, thus, may provide a potential application in tissue engineering of bone tissue.

How pollen tubes grow.

Sexual reproduction of flowering plants depends on delivery of the sperm to the egg, which occurs through a long, polarized projection of a pollen cell, called the pollen tube. The pollen tube grows exclusively at its tip, and this growth is distinguished by very fast rates and reaches extended lengths. Thus, one of the most fascinating aspects of pollen biology is the question of how enough cell wall material is produced to accommodate such rapid extension of pollen tube, and how the cell wall deposition and structure are regulated to allow for rapid changes in the direction of growth. This review discusses recent advances in our understanding of the mechanism of pollen tube growth, focusing on such basic cellular processes as control of cell shape and growth by a network of cell wall-modifying enzymes, molecular motor-mediated vesicular transport, and intracellular signaling by localized gradients of second messengers.

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.

Pollen-specific pectin methylesterase involved in pollen tube growth.

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.

Effects of calreticulin on viral cell-to-cell movement.

Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.

Signal peptide-dependent targeting of a rice alpha-amylase and cargo proteins to plastids and extracellular compartments of plant cells.

alpha-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether alpha-amylases are localized in plastids where starch is stored. To study the subcellular localization of alpha-amylases in plant cells, a rice (Oryza sativa) alpha-amylase, alphaAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of alphaAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated alphaAmy3 to be cotranslationally imported and processed in canine microsomes. alphaAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with Mr similar to alphaAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized alphaAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the alphaAmy3 SP was further studied by gain-of-function analyses. beta-Glucuronidase (GUS) and green fluorescence protein fused with or without the alphaAmy3 SP was expressed in transgenic tobacco or rice. The alphaAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice alpha-amylase, alphaAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.

Systemic movement of a tobamovirus requires host cell pectin methylesterase.

Systemic movement of plant viruses through the host vasculature, one of the central events of the infection process, is essential for maximal viral accumulation and development of disease symptoms. The host plant proteins involved in this transport, however, remain unknown. Here, we examined whether or not pectin methylesterase (PME), one of the few cellular proteins known to be involved in local, cell-to-cell movement of tobacco mosaic virus (TMV), is also required for the systemic spread of viral infection through the plant vascular system. In a reverse genetics approach, PME levels were reduced in tobacco plants using antisense suppression. The resulting PME antisense plants displayed a significant degree of PME suppression in their vascular tissues but retained the wild-type pattern of phloem loading and unloading of a fluorescent solute. Systemic transport of TMV in these plants, however, was substantially delayed as compared to the wild-type tobacco, suggesting a role for PME in TMV systemic infection. Our analysis of virus distribution in the PME antisense plants suggested that TMV systemic movement may be a polar process in which the virions enter and exit the vascular system by two different mechanisms, and it is the viral exit out of the vascular system that involves PME.

A novel plant homeodomain protein interacts in a functionally relevant manner with a virus movement protein.

Tomato bushy stunt virus and its cell-to-cell movement protein (MP; P22) provide valuable tools to study trafficking of macromolecules through plants. This study shows that wild-type P22 and selected movement-defective P22 amino acid substitution mutants were equivalent for biochemical features commonly associated with MPs (i.e. RNA binding, phosphorylation, and membrane partitioning). This generated the hypothesis that their movement defect was caused by improper interaction between the P22 mutants and one or more host factors. To test this, P22 was used as bait in a yeast (Saccharomyces cerevisiae) two-hybrid screen with a tobacco (Nicotiana tabacum) cDNA library, which identified a new plant homeodomain leucine-zipper protein that reproducibly interacted with P22 but not with various control proteins. These results were confirmed with an independent in vitro binding test. An mRNA for the host protein was detected in plants, and its accumulation was enhanced upon Tomato bushy stunt virus infection of two plant species. The significance of this interaction was further demonstrated by the failure of the homeodomain protein to interact efficiently with two of the well-defined movement-deficient P22 mutants in yeast and in vitro. This is the first report, to our knowledge, that a new plant homeodomain leucine-zipper protein interacts specifically and in a functionally relevant manner with a plant virus MP.