Journal: BMC Cell Biology

Article Title: Human ASPL/TUG interacts with p97 and complements the proteasome mislocalization of a yeast ubx4 mutant, but not the ER-associated degradation defect

doi: 10.1186/1471-2121-15-31

Figure Lengend Snippet: ASPL interacts with p97 via the UBX domain. (a) Yeast two-hybrid analyses of p97 using the HIS3 reporter gene. Co-transformation of p97 bait with the indicated p97 binding partner preys supported cell growth under conditions selecting for interaction (in the absence of histidine and the presence of 25 mM 3-aminotriazol (3AT)) (right panel). An empty prey vector served as a negative control. (b) Yeast two-hybrid analyses of ASPL using the HIS3 reporter gene. Co-transformation of ASPL bait with p97 or NSF preys supported cell growth under conditions selecting for interaction (in the absence of histidine and the presence of 25 mM 3-aminotriazol (3AT)) (right panel). An empty prey vector served as a negative control. (c) Purified 6His-tagged p97 was incubated with GST or GST-tagged ASPL and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to p97 (upper panel) Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (d) Purified 6His-tagged NSF was incubated with GST or GST-tagged ASPL and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to the 6His-tag on NSF (upper panels). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). Interaction to NSF was only evident when no detergents were included in the buffer system. In the presence of 0.5% Triton X-100 no interaction between ASPL and NSF was observed. (e) MelJuSo cell lysates were used in immunoprecipitation (IP) experiments with antibodies to ASPL and Protein A Sepharose or as a control Protein A Sepharose beads only. SDS-PAGE and blotting revealed that ASPL co-precipitated p97, but not NSF or the Rpn1 or α subunits of the 26S proteasome.

Article Snippet: The expression constructs for human p97 were kindly provided by Prof. Hemmo H. Meyer (Zürich, Switzerland).

Techniques: Transformation Assay, Binding Assay, Plasmid Preparation, Negative Control, Purification, Incubation, SDS Page, Staining, Immunoprecipitation, Control

Journal: BMC Cell Biology

Article Title: Human ASPL/TUG interacts with p97 and complements the proteasome mislocalization of a yeast ubx4 mutant, but not the ER-associated degradation defect

doi: 10.1186/1471-2121-15-31

Figure Lengend Snippet: ASPL interacts with the p97 N-domain. (a) Schematic diagram of the ASPL domain organization and the various truncations used in the precipitation experiments. (b) Purified 6His-tagged p97 was incubated with GST or the indicated GST-tagged ASPL truncations and precipitated with glutathione (GSH) Sepharose. Bound proteins were analyzed by SDS-PAGE and blotting using antibodies to the 6His-tag on p97 (upper panel). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel). (c) Schematic diagram of the p97 domain organization and the various truncations used in the precipitation experiments. (d) Purified 6His-tagged p97 and p97 truncations were incubated with GST and GST-tagged ASPL before precipitation and analysis by SDS-PAGE and blotting using antibodies specific for the 6His-tagged p97 proteins (upper panel). Even loading was checked by staining with Coomassie Brilliant Blue (CBB) (lower panel).

Article Snippet: The expression constructs for human p97 were kindly provided by Prof. Hemmo H. Meyer (Zürich, Switzerland).

Techniques: Purification, Incubation, SDS Page, Staining

Journal: The Journal of General Physiology

Article Title: Regulation of AQP0 water permeability is enhanced by cooperativity

doi: 10.1085/jgp.201210884

Figure Lengend Snippet: Topology and structure of AQP0. (A) Each AQP0 monomer is composed of six transmembrane domains (1–6) and five loops (A–E) in rainbow color (ROYGIBV). The pore is formed by two half helices and loops B and E (in green) folded back into the membrane. Major players in the cooperativity are H40 in the external loop A and S235 in the C terminus tail. (B) The tetramer is shown from the extracelluar side with the four monomers (blue and mustard) and the two CaMs (red). Both panels were generated with VMD from a molecular dynamics simulation based on crystal structures (deposited in the Protein Data Bank under accession no. 1TM8 for AQP0; ; and Protein Data Bank accession no. 1NWD for CaM; ) originally modeled together in .

Article Snippet: The expression construct for bovine AQP0 (pXβG-AQP0) was provided by P. Agre (Johns Hopkins University, Baltimore, MD).

Techniques: Membrane, Generated

Journal: The Journal of General Physiology

Article Title: Regulation of AQP0 water permeability is enhanced by cooperativity

doi: 10.1085/jgp.201210884

Figure Lengend Snippet: pH dose–response curve of the water permeability of AQP0. The solid black line is a fit to the Hill equation, with n = 4 (χ 2 /DOF = 0.0068 and R 2 = 0.994).

Article Snippet: The expression construct for bovine AQP0 (pXβG-AQP0) was provided by P. Agre (Johns Hopkins University, Baltimore, MD).

Techniques: Permeability

Journal: The Journal of General Physiology

Article Title: Regulation of AQP0 water permeability is enhanced by cooperativity

doi: 10.1085/jgp.201210884

Figure Lengend Snippet: Calcium cooperativity. (A) Effect of calcium concentration on the water permeability of mixed injection of AQP0 (WT) and a mutant S235D. Mix 1:1 represents 10 ng AQP0 and 10 ng of mutant H40C (fraction of insensitive monomer = 10/20 = 0.5), mix 1:5 represents 2 ng AQP0 and 10 ng of mutant H40C (fraction of insensitive monomer = 2/12 = 0.17), mix 5:1 = 10 ng AQP0 and 2 ng of mutant H40C (fraction of insensitive monomer = 2/12 = 0.17), and mix 1:9 represents 1 ng AQP0 and 9 ng S235D. The left-hand panel shows the response of WT AQP0. Note that P f increases when the calcium concentration is decreased. In contrast, an increased calcium concentration increases P f in the S235D mutant. When the two are mixed so as to form hetero tetramers, even 20% of WT is sufficient to suppress completely the P f increase induced by 5 mM calcium, whereas the proportional increase in P f induced by lowering calcium remains. When the mixture contains 80% WT, the low calcium response is still present in proportion to the amount of WT, but the elevated calcium response is completely suppressed. The horizontal dotted line represents the water permeability of uninjected oocytes. (B) Fraction of increase plotted against the fraction of insensitive monomer. (For no Ca 2+ response, S235D is the insensitive monomer. For 5-mM Ca 2+ response, WT is the insensitive monomer.) Experimental results for 5-mM Ca 2+ increase are plotted as purple triangles and are well fit by a curve calculated from the binomial distribution assuming one insensitive monomer is sufficient to render the whole tetramer insensitive and with a bias of 0.5 kT for WT to associate with mutants in a tetramer (dashed purple curve; χ 2 = 1.260). The dotted black curve is calculated assuming no bias and that one insensitive monomer is sufficient to render the entire tetramer insensitive to Ca 2+ increase (χ 2 = 3.133). Experimental data for zero calcium are plotted as blue squares and are well fit by a straight line (the theoretical prediction assuming each monomer acts independently of the others in the tetramer; χ 2 /DOF = 0.18774 and R 2 = 0.557; blue straight line). Positive and negative errors are equal, but error bars are shown in only one direction to avoid clutter.

Article Snippet: The expression construct for bovine AQP0 (pXβG-AQP0) was provided by P. Agre (Johns Hopkins University, Baltimore, MD).

Techniques: Concentration Assay, Permeability, Injection, Mutagenesis

Journal: The Journal of General Physiology

Article Title: Regulation of AQP0 water permeability is enhanced by cooperativity

doi: 10.1085/jgp.201210884

Figure Lengend Snippet: pH cooperativity. (A) Effect of acid and alkaline pH on the water permeability of mixtures of AQP0 (WT) and the H40C mutant. WT responds to acid pH, whereas the H40C mutant responds to alkaline pH. Mix 5:1 represents 10 ng AQP0 and 2 ng of mutant H40C (fraction of insensitive monomer = 2/12 = 0.166), and mix 1:1 represents 10 ng AQP0 and 10 ng of mutant H40C (fraction of insensitive monomer = 10/20 = 0.5). The P f of the mix 1:1 at pH 8.5 is greater than the P f at pH 7.5, with a p-value of 5 × 10 −5 (Student’s t test). The horizontal dotted line represents the water permeability of uninjected oocytes. (B) Fraction of increase plotted against the fraction of insensitive monomer. (For acid pH, the H40C mutant is the insensitive monomer. For alkaline pH, WT is the insensitive monomer.) Experimental results for acid pH are plotted as red squares and are well fit by a curve calculated from the binomial distribution of monomers randomly partitioning into each tetramer and the assumption that a single insensitive monomer renders the whole tetramer insensitive to acid pH (dashed red line; χ 2 = 0.840). The curve assuming that two insensitive monomers are required to render the tetramer insensitive to acid pH (dashed black line; χ 2 = 8.88) does not fit the experimental data. Experimental results for alkaline pH are plotted as green circles and are well fit by a straight line (the theoretical prediction assuming each monomer acts independently of the others in the tetramer; χ 2 /DOF = 0.071 and R 2 = 0.910; solid green line). Each data point is the average of experiments using nine oocytes from three different batches.

Article Snippet: The expression construct for bovine AQP0 (pXβG-AQP0) was provided by P. Agre (Johns Hopkins University, Baltimore, MD).

Techniques: Permeability, Mutagenesis