A Bni4-Glc7 phosphatase complex that recruits chitin synthase to the site of bud emergence.

Bni4 is a scaffold protein in the yeast Saccharomyces cerevisiae that tethers chitin synthase III to the bud neck by interacting with septin neck filaments and with Chs4, a regulatory subunit of chitin synthase III. We show herein that Bni4 is also a limiting determinant for the targeting of the type 1 serine/threonine phosphatase (Glc7) to the bud neck. Yeast cells containing a Bni4 variant that fails to associate with Glc7 fail to tether Chs4 to the neck, due in part to the failure of Bni4(V831A/F833A) to localize properly. Conversely, the Glc7-129 mutant protein fails to bind Bni4 properly and glc7-129 mutants exhibit reduced levels of Bni4 at the bud neck. Bni4 is phosphorylated in a cell cycle-dependent manner and Bni4(V831A/F833A) is both hyperphosphorylated and mislocalized in vivo. Yeast cells lacking the protein kinase Hsl1 exhibit increased levels of Bni4-GFP at the bud neck. GFP-Chs4 does not accumulate at the incipient bud site in either a bni4::TRP1 or a bni4(V831A/F833A) mutant but does mobilize to the neck at cytokinesis. Together, these results indicate that the formation of the Bni4-Glc7 complex is required for localization to the site of bud emergence and for subsequent targeting of chitin synthase.

Oxidative stress promotes degradation of the Irr protein to regulate haem biosynthesis in Bradyrhizobium japonicum.

The haem proteins catalase and peroxidase are stress response proteins that detoxify reactive oxygen species. In the bacterium Bradyrhizobium japonicum, expression of the gene encoding the haem biosynthesis enzyme delta-aminolevulinic acid dehydratase (ALAD) is normally repressed by the Irr protein in iron-limited cells. Irr degrades in the presence of iron, which requires haem binding to the protein. Here, we found that ALAD levels were elevated in iron-limited cells of a catalase-deficient mutant, which corresponded with aberrantly low levels of Irr. Irr was undetectable in wild-type cells within 90 min after exposure to exogenous H2O2, but not in a haem-deficient mutant strain. In addition, Irr did not degrade in response to iron in the absence of O2. The findings indicate that reactive oxygen species promote Irr turnover mediated by haem, and are involved in iron-dependent degradation. We demonstrated Irr oxidation in vitro, which required haem, O2 and a reductant. A truncated Irr mutant unable to bind ferrous haem does not degrade in vivo, and was not oxidized in vitro. We suggest that Irr oxidation is a signal for its degradation, and that cells sense and respond to oxidative stress through Irr to regulate haem biosynthesis.

KatG is the primary detoxifier of hydrogen peroxide produced by aerobic metabolism in Bradyrhizobium japonicum.

Bacteria are exposed to reactive oxygen species from the environment and from those generated by aerobic metabolism. Catalases are heme proteins that detoxify H(2)O(2), and many bacteria contain more than one catalase enzyme. Also, the nonheme peroxidase alkyl hydroperoxide reductase (Ahp) is the major scavenger of endogenous H(2)O(2) in Escherichia coli. Here, we show that aerobically grown Bradyrhizobium japonicum cells express a single catalase activity. Four genes encoding putative catalases in the B. japonicum genome were identified, including a katG homolog encoding a catalase-peroxidase. Deletion of the katG gene resulted in loss of catalase activity in cell extracts and of exogenous H(2)O(2) consumption by whole cells. The katG strain had a severe aerobic growth phenotype but showed improved growth in the absence of O(2). By contrast, a B. japonicum ahpCD mutant grew well aerobically and consumed H(2)O(2) at wild-type rates. A heme-deficient hemA mutant expressed about one-third of the KatG activity as the wild type but grew well aerobically and scavenged low concentrations of exogenous H(2)O(2). However, cells of the hemA strain were deficient in consumption of high concentrations of H(2)O(2) and were very sensitive to killing by short exposure to H(2)O(2). In addition, KatG activity did not decrease as a result of mutation of the gene encoding the transcriptional activator OxyR. We conclude that aerobic metabolism produces toxic levels of H(2)O(2) in B. japonicum, which is detoxified primarily by KatG. Furthermore, the katG level sufficient for detoxification does not require OxyR.

Plasma membrane localization of the Yck2p yeast casein kinase 1 isoform requires the C-terminal extension and secretory pathway function.

The S. cerevisiae Yck2 protein is a plasma membrane-associated member of the casein kinase 1 protein kinase family that, with its homolog Yck1p, is required for bud morphogenesis, cytokinesis, endocytosis and other cellular processes. Membrane localization of Yckp is critical for its function, since soluble mutants do not provide sufficient biological activity to sustain normal growth. Yck2p has neither a predicted signal sequence nor obvious transmembrane domain to achieve its plasma membrane localization, but has a C-terminal -Cys-Cys sequence that is likely to be palmitoylated. We demonstrate here that Yck2p is targeted through association with vesicular intermediates of the classical secretory pathway. Yck2p lacking C-terminal Cys residues fails to associate with any membrane, whereas substitution of these residues with a farnesyl transferase signal sequence allows sec-dependent plasma membrane targeting and biological function, suggesting that modification is required for interaction with early secretory membranes but that targeting does not require a particular modification. Deletion analysis within the 185 residue C-terminus indicates that the final 28 residues are critical for membrane association, and additional sequences just upstream are required for proper plasma membrane targeting.