Plasma membrane proteins internalized by endocytosis and targeted for degradation are sorted into lumenal vesicles of multivesicular bodies (MVBs) by the endosomal sorting complexes required for transport (ESCRT) machinery. Here, we show that the Arabidopsis thaliana ESCRT-related CHARGED MULTIVESICULAR BODY PROTEIN/CHROMATIN MODIFYING PROTEIN1A (CHMP1A) and CHMP1B proteins are essential for embryo and seedling development. Double homozygous chmp1a chmp1b mutant embryos showed limited polar differentiation and failed to establish bilateral symmetry. Mutant seedlings show disorganized apical meristems and rudimentary true leaves with clustered stomata and abnormal vein patterns. Mutant embryos failed to establish normal auxin gradients. Three proteins involved in auxin transport, PINFORMED1 (PIN1), PIN2, and AUXIN-RESISTANT1 (AUX1) mislocalized to the vacuolar membrane of the mutant. PIN1 was detected in MVB lumenal vesicles of control cells but remained in the limiting membrane of chmp1a chmp1b MVBs. The chmp1a chmp1b mutant forms significantly fewer MVB lumenal vesicles than the wild type. Furthermore, CHMP1A interacts in vitro with the ESCRT-related proteins At SKD1 and At LIP5. Thus, Arabidopsis CHMP1A and B are ESCRT-related proteins with conserved endosomal functions, and the auxin carriers PIN1, PIN2, and AUX1 are ESCRT cargo proteins in the MVB sorting pathway.

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, a-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zeinrich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.The cereal endosperm consists of three main cell types: an inner mass of starchy endosperm cells, one to three layers of epidermal aleurone cells, and the transfer cells that contact the maternal vascular tissue (Olsen, 2004). The starchy endosperm accounts for 80 to 90% of the grain weight and contains large amounts of storage proteins and starch. These cells undergo programmed cell death during maturation. Aleurone cells are rich in protein storage vacuoles (PSVs), minerals, and lipid bodies and remain alive during seed development. It is assumed that the breakdown of proteins localized to PSVs in aleurone cells provides an essential source of the amino acids necessary for the synthesis of hydrolytic enzymes required for mobilizing food stored in the starchy endosperm (Filner and Varner, 1967;Jacobsen et al., 1988;Bethke et al., 1998).Besides its biological relevance as a model to study plant development, cell differentiation, and programmed cell death, the cereal endosperm is very important in terms of its nutritional value. Cereal grains contain less protein than do legume seeds, but because cereals are produced and consumed in much larger quantities, they are the main source of protein for the nutrition of humans and livestock (Shewry and Halford, 2002). The major storage proteins in maize (Zea mays) kernels are the alcoholsoluble prolamins, a type of storage proteins present only in grasses. Maize prolamins, referred to as zeins, are divided into different types: a-, b-, g-, and d-zeins (Coleman and Larkins, 1999) that differ in amino acid composition and structural properties (Shewry and Halford, 2002)...

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The phototrophic bacterium Rhodobacter sphaeroides DSM 158 is able to reduce nitrate to nitrite by means of a periplasmic nitrate reductase which is induced by nitrate and is not repressed by ammonium or oxygen. Recently, a 6.8 kb PstI DNA fragment carrying the napABC genes coding for this periplasmic nitrate-reducing system was cloned [Reyes, Roldán, Klipp, Castillo and Moreno-Vivián (1996) Mol. Microbiol. 19, 1307-1318]. Further sequence and genetic analyses of the DNA region upstream from the napABC genes reveal the presence of four additional nap genes. All these R. sphaeroides genes seem to be organized into a napKEFDABC transcriptional unit. In addition, a partial open reading frame similar to the Azorhizobium caulinodans yntC gene and the Escherichia coli yjcC and yhjK genes is present upstream from this nap gene cluster. The R. sphaeroides napK gene codes for a putative 6.3 kDa transmembrane protein which is not similar to known proteins and the napE gene codes for a 6.7 kDa transmembrane protein similar to the Thiosphaera pantotropha NapE. The R. sphaeroides napF gene product is a 16.4 kDa protein with four cysteine clusters that probably bind four [4Fe-4S] centres. This iron-sulphur protein shows similarity to the NapF and NapG proteins of E. coli and Haemophilus influenzae. Finally, the napD gene product is a 9.4 kDa soluble protein which is also found in E. coli and T. pantotropha. The 5' end of the nap transcript has been determined by primer extension, and a sigma70-like promoter has been identified upstream from the napK gene. The same transcriptional start site is found for cells growing aerobically or anaerobically with nitrate. Different mutant strains carrying defined polar and non-polar insertions in each nap gene were constructed. Characterization of these mutant strains demonstrates the participation of the nap gene products in the periplasmic nitrate reduction in R. sphaeroides.

The phototrophic bacterium Rhodobacter sphaeroides DSM 158 has a periplasmic nitrate reductase which is induced by nitrate and it is not repressed by ammonium or oxygen. In a Tn5 mutant lacking nitrate reductase activity, transposon insertion is localized in a 1.2 kb EcoRI fragment. A 0.6 kb BamHI-EcoRI segment of this region was used as a probe to isolate, from the wild-type strain, a 6.8 kb PstI fragment carrying the putative genes coding for the periplasmic nitrate reductase. In vivo protein expression and DNA sequence analysis reveal the presence in this region of three genes, napABC, probably organized in an operon. These genes are required for nitrate reduction, as deduced by mutational and complementation studies. The napA gene codes for a protein with a high homology to the periplasmic nitrate reductase from Alcaligenes eutrophus and, to a lesser extent, to other prokaryotic nitrate reductases and molybdenum-containing enzymes. The napB gene product has two haem c-binding sites and shows a high homology with the cytochrome c-type subunit of the periplasmic nitrate reductase from A. eutrophus. NAPA and NAPB proteins appear to be translated with signal peptides of 29 and 24 residues, respectively, indicating that mature proteins are located in the periplasm. The napC gene codes for a 25 kDa protein with a transmembrane sequence of 17 hydrophobic residues. NAPC has four haem c-binding sites and is homologous to the membrane-bound c-type cytochromes encoded by Pseudomonas stutzeri nirT and Escherichia coli torC genes. The phenotypes of defined insertion mutants constructed for each gene also indicate that periplasmic nitrate reductase from R. sphaeroides DSM 158 is a dimeric complex of a 90 kDa catalytic subunit (NAPA) and a 15 kDa cytochrome c (NAPB), which receives electrons from a membrane-anchored tetrahaem protein (NAPC), thus allowing electron flow between membrane and periplasm. This nitrate-reducing system differs from the assimilatory and respiratory bacterial nitrate reductases at the level of cellular localization, regulatory properties, biochemical characteristics and gene organization.

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The molecular mechanisms by which vascular tissues acquire their identities are largely unknown. Here, we report on the identification and characterization of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC), a member of a 15-member, plant-specific gene family in Arabidopsis (Arabidopsis thaliana) that encodes proteins of unknown function with four predicted transmembrane domains. Homozygous vcc mutants displayed cotyledon vein networks of reduced complexity and disconnected veins. Similar disconnections or gaps were observed in the provasculature of vcc embryos, indicating that defects in vein connectivity appear early in mutant embryo development. Consistently, the overexpression of VCC leads to an unusually high proportion of cotyledons with high-complexity vein networks. Neither auxin distribution nor the polar localization of the auxin efflux carrier were affected in vcc mutant embryos. Expression of VCC was detected in developing embryos and procambial, cambial, and vascular cells of cotyledons, leaves, roots, hypocotyls, and anthers. To evaluate possible genetic interactions with other genes that control vasculature patterning in embryos, we generated a double mutant for VCC and OCTOPUS (OPS). The vcc ops double mutant embryos showed a complete loss of high-complexity vascular networks in cotyledons and a drastic increase in both provascular and vascular disconnections. In addition, VCC and OPS interact physically, suggesting that VCC and OPS are part of a complex that controls cotyledon vascular complexity.