SummaryThe AtSTP2 gene (sugar transport protein 2) of Arabidopsis thaliana encodes a high affinity, low specificity monosaccharide carrier that can transport a number of hexoses and pentoses at similar rates. AtSTP2 has 12 putative transmembrane helices and a molecular mass of 55.0 kDa. AtSTP2 expression was localized in AtSTP2 promoter-β-glucuronidase (GUS) Arabidopsis plants showing AtSTP2-driven GUS activity during pollen maturation and also in germinating pollen. Immunohistochemical studies with anti-AtSTP2 antiserum as well as RNA in situ hybridization analyses modified these results and showed that AtSTP2 expression is confined to the early stages of gametophyte development. Both AtSTP2 mRNA and AtSTP2 protein are first seen at the time of beginning callose degradation and microspore release from the tetrades. AtSTP2 mRNA and AtSTP2 protein are no longer detected after the mitotic divisions and the formation of the trinucleate gametophyte. No AtSTP2 mRNA or AtSTP2 protein is seen in fully developed or germinating pollen. The putative role of AtSTP2 in the uptake of glucose units resulting from callose degradation during pollen maturation is discussed.

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi-l-arginyl-poly-l-aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25±30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.

When cyanobacteria are starved for nitrogen, expression of the NblA protein increases and thereby induces proteolytic degradation of phycobilisomes, light-harvesting complexes of pigmented proteins. Phycobilisome degradation leads to a color change of the cells from blue-green to yellow-green, referred to as bleaching or chlorosis. As reported previously, NblA binds via a conserved region at its C terminus to the ␣-subunits of phycobiliproteins, the main components of phycobilisomes. We demonstrate here that a highly conserved stretch of amino acids in the N-terminal helix of NblA is essential for protein function in vivo. Affinity purification of glutathione S-transferase-tagged NblA, expressed in a Nostoc sp. PCC7120 mutant lacking wildtype NblA, resulted in co-precipitation of ClpC, encoded by open reading frame alr2999 of the Nostoc chromosome. ClpC is a HSP100 chaperone partner of the Clp protease. ATP-dependent binding of NblA to ClpC was corroborated by in vitro pulldown assays. Introducing amino acid exchanges, we verified that the conserved N-terminal motif of NblA mediates the interaction with ClpC. Further results indicate that NblA binds phycobiliprotein subunits and ClpC simultaneously, thus bringing the proteins into close proximity. Altogether these results suggest that NblA may act as an adaptor protein that guides a ClpC⅐ClpP complex to the phycobiliprotein disks in the rods of phycobilisomes, thereby initiating the degradation process.

Cyanobacterial light-harvesting complexes, the phycobilisomes, are proteolytically degraded when the organisms are starved for combined nitrogen, a process referred to as chlorosis or bleaching. Gene nblA, present in all phycobilisome-containing organisms, encodes a protein of about 7 kDa that plays a key role in phycobilisome degradation. The mode of action of NblA in this degradation process is poorly understood. Here we presented the 1.8-Å crystal structure of NblA from Anabaena sp. PCC 7120. In the crystal, NblA is present as a four-helix bundle formed by dimers, the basic structural units. By using pull-down assays with immobilized NblA and peptide scanning, we showed that NblA specifically binds to the ␣-subunits of phycocyanin and phycoerythrocyanin, the main building blocks of the phycobilisome rod structure. By site-directed mutagenesis, we identified amino acid residues in NblA that are involved in phycobilisome binding. The results provided evidence that NblA is directly involved in phycobilisome degradation, and the results allowed us to present a model that gives insight into the interaction of this small protein with the phycobilisomes.Cyanobacteria are photosynthetic prokaryotes, which perform planttype oxygenic photosynthesis. Light harvesting in these organisms is mainly mediated by phycobilisomes, large water-soluble multiprotein complexes that are attached to the cytoplasmic surface of the photosynthetic membrane. Phycobilisomes are composed of brilliantly colored phycobiliproteins, to which open chain tetrapyrrole chromophores are covalently attached, and of linker proteins, which, with one

This paper describes the expression analyses of the