A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM spans and are possibly linked to transport functions. The ARAMEMNON DB enables direct comparison of the predictions of seven different TM span computation programs and the predictions of subcellular localization by eight signal peptide recognition programs. A special function displays the proteins related to the query and dynamically generates a protein family structure. As a first set of proteins from other organisms, all of the approximately 700 putative membrane proteins were extracted from the genome of the cyanobacterium Synechocystis sp. and incorporated in the ARAMEMNON DB. The ARAMEMNON DB is accessible at the URL http://aramemnon.botanik.uni-koeln.de.Biological membranes constitute a chemical barrier to the environment and are thus the prerequisite for the establishment and maintenance of a controlled intracellular milieu, the cytoplasm. In eukaryotes, membranes are also responsible for the formation of chemically distinct intracellular compartments. The lipid bilayer membranes contain a great diversity of proteins that fulfill different functions and serve as an interface to the environment and between different compartments. Among these membrane proteins are receptors involved in signaling cascades and pathogen defense reactions, enzymes such as the apparatus for cell wall biosynthesis, and transporters responsible for the import and export of solutes and ions and the establishment of electrochemical gradients across membranes, thereby connecting the different metabolic pathways of the cellular compartments and organelles.Many plant transport proteins were identified by complementation of yeast mutants that were deficient in certain transport or metabolic functions (Frommer and Ninnemann, 1995). Membrane proteins have a modular structure, consisting of hydrophobic domains and hydrophilic loops or termini that extend into the cytoplasm, the organelle, or point to the extracellular space. The hydrophobic transmembrane (TM) domains consist of amphipathic ␣-helices or ␤-barrels that pass across or dip into the hydrophobic membrane lipid bilayer. During recent years, the three-dimensional structures of more than 160 TM proteins or domains were determined at varying resolution, and it appears that modularity is a general feature of polytopic membrane proteins (http://www.rcsb.org/pdb/; http://www.ncbi.nlm. nih.gov:80/Structure/; Berman et al., 2002;.Arabidopsis is the first plant for which the genome has been deciphered...

Protective immunity against Mycobacterium tuberculosis involves major histocompatibility complex class I (MHC-I)- and CD1-restricted CD8 T cells, but the mechanisms underlying antigen delivery to antigen-presenting molecules remain enigmatic. Macrophages, the primary host cells for mycobacteria, are CD1-negative. Here we show that M. tuberculosis phagosomes are secluded from the cytosolic MHC-I processing pathway and that mycobacteria-infected cells lose their antigen-presenting capacity. We also show that mycobacteria induce apoptosis in macrophages, causing the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen-presenting cells (APCs). Inhibition of apoptosis reduced transfer of antigens to bystander cells and activation of CD8 T cells. Uninfected dendritic cells, which engulfed extracellular vesicles, were indispensable for subsequent cross-presentation of antigens, through MHC-I and CD1b, to T cells from mycobacteria-sensitized donors. This new 'detour' pathway for presentation of antigens from a phagosome-contained pathogen shows the functional significance of infection-induced apoptosis in the activation of CD8 T cells specific for both protein and glycolipid antigens in tuberculosis.

A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-␥ production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d. I n contrast to classical MHC molecules, the nonpolymorphic CD1 proteins present lipid antigens to T cells (1). These evolutionaryconserved antigen-presenting molecules are divided into group I (consisting of CD1a, CD1b, CD1c, and CD1e in humans) and group II (represented by CD1d in mice and humans) (2, 3). CD1 molecules are 43-to 49-kDa cell-surface glycoproteins homologous to MHC class I molecules with a limited allelic polymorphism (4). Compared with MHC class I, they possess a deeper and more hydrophobic antigen-binding groove. Human CD1a, CD1b, and CD1c present mammalian and mycobacterial lipids to CD4 and CD8 T cells (2, 5). CD1d-restricted T cells appear to be primarily self-reactive, and they have been implicated in the control of autoimmune diseases (6, 7). The marine sponge-derived lipid ␣-galactosylceramide (␣-GalCer), in the context of CD1d, is a potent stimulator of all V␣14-J␣281 T cell receptor (TCR)-expressing natural killer T cells (NKT cells) in mice and their cognates in humans expressing V␣24-J␣Q TCR. Therefore, although ␣-GalCer is an artificial ligand of unclear physiological relevance, this lipid is a useful tool to study CD1d-restricted NKT cells in mammals (8, 9). The NKT cell subset is considered to perform regulatory, rather than host-defense, functions with the following two self antigens identified so far: phosphatidylinositol (PI) and the tumor-associated disialoganglioside GD3 (10, 11). To our knowledge, no bacterial antigen has been identified, which is presented by CD1d.In analyzing the structural determinants of mycobacterial and mammalian lipids for binding to CD1d and recognition by T cells, we identified a PI mannoside (PIM), which induced IFN-␥ release and cytotoxicity in a CD1d-restricted manner, from the mycobacterial cell wall. Hence, this PIM is a bacterial antigen for human and murine NKT cells. Materials and MethodsChemicals. All reagents were purchased from Sigma, unless indicated otherwise. ␣-GalCer was kindly provided by Pharmaceutical Research Laboratories (Kirin Brewery, Gumna, Japan).Mice. All mice were bred and housed under specific pat...

Plastids of nongreen tissues can import carbon in the form of glucose 6-phosphate via the glucose 6-phosphate/phosphate translocator (GPT). The Arabidopsis thaliana genome contains two homologous GPT genes, AtGPT1 and AtGPT2. Both proteins show glucose 6-phosphate translocator activity after reconstitution in liposomes, and each of them can rescue the low-starch leaf phenotype of the pgi1 mutant (which lacks plastid phosphoglucoisomerase), indicating that the two proteins are also functional in planta. AtGPT1 transcripts are ubiquitously expressed during plant development, with highest expression in stamens, whereas AtGPT2 expression is restricted to a few tissues, including senescing leaves. Disruption of GPT2 has no obvious effect on growth and development under greenhouse conditions, whereas the mutations gpt1-1 and gpt1-2 are lethal. In both gpt1 lines, distorted segregation ratios, reduced efficiency of transmission in males and females, and inability to complete pollen and ovule development were observed, indicating profound defects in gametogenesis. Embryo sac development is arrested in the gpt1 mutants at a stage before the fusion of the polar nuclei. Mutant pollen development is associated with reduced formation of lipid bodies and small vesicles and the disappearance of dispersed vacuoles, which results in disintegration of the pollen structure. Taken together, our results indicate that GPT1-mediated import of glucose 6-phosphate into nongreen plastids is crucial for gametophyte development. We suggest that loss of GPT1 function results in disruption of the oxidative pentose phosphate cycle, which in turn affects fatty acid biosynthesis.