Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic “leucine collar” that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate.     

The inner-mitochondrial distribution of Oxa1 depends on the growth conditions and on the availability of substrates

The Oxa1 protein is a well-conserved integral protein of the inner membrane of mitochondria. It mediates the insertion of both mitochondrial- and nuclear-encoded proteins from the matrix into the inner membrane. We investigated the distribution of budding yeast Oxa1 between the two subdomains of the contiguous inner membrane--the cristae membrane (CM) and the inner boundary membrane (IBM)--under different physiological conditions. We found that under fermentable growth conditions, Oxa1 is enriched in the IBM, whereas under nonfermentable (respiratory) growth conditions, it is predominantly localized in the CM. The enrichment of Oxa1 in the CM requires mitochondrial translation; similarly, deletion of the ribosome-binding domain of Oxa1 prevents an enrichment of Oxa1 in the CM. The predominant localization in the IBM under fermentable growth conditions is prevented by inhibiting mitochondrial protein import. Furthermore, overexpression of the nuclear-encoded Oxa1 substrate Mdl1 shifts the distribution of Oxa1 toward the IBM. Apparently, the availability of nuclear- and mitochondrial-encoded substrates influences the inner-membrane distribution of Oxa1. Our findings show that the distribution of Oxa1 within the inner membrane is dynamic and adapts to different physiological needs.     

Das Vogelleben in Flur und Wald des deutsch-b?hmischen Mittelgebirges

Ohne Zusammenfassung     

Concanavalin A induced changes of lymphocyte p-nitrophenylphosphatase (author's transl)]

The effect of K+, Na+, Mg2+ and ATP on the p-nitrophenylphosphatase activity was investigated. As an enzyme preparation a microsomal fraction of sheep lymphocytes was used. Low concentrations of Mg2+, K+ and Na+ increased, whereas high concentrations decreased the enzyme activity. There was an inhibition of activity by ATP without Na+ in the incubation medium and an increase of enzyme activity at low K:Na-ratio. By concanavalin A in a concentration of 15 mug/ml the p-nitrophenylphosphatase activity was increased in intact cells and the microsomal fraction for 30-40%. The activation was not Na+, K+, Mg2+, p-nitrophenylphosphate or ATP dependent.     

Temperature-dependence of the proliferation of mouse bone marrow cells cultured in H2O- and D2O-media.

In an attempto to optimize and standardize the in vitro culture conditions of mouse bone marrow cells for assaying growth regulating factors, we studied the effects of incubation at low temperatures and of a nutrient medium containing deuteriumoxide instead of water. It was found that (1) the proliferative capacity of the cells is significantly increased by pre-incubation for 1-2 h at 0 degrees C rather than at 37 degrees C, measured by both a colony-forming and a 3H-thymidine (3H-tdr) uptake assay. A similar temperature effect on the colony-forming and 3H-tdr uptake ability is apparent after pre-incubation in D2O-medium, yet significantly lower than in H2O-medium. It was concluded that the previously observed protective effects of D2O on ascites tumor cell proliferation and viability and for hemolysis of human erythrocytes is not apparent in proliferating and colony-forming mouse bone marrow cells in vitro.