Conformational fluctuations of the Ca2+-ATPase in the native membrane environment. Effects of pH, temperature, catalytic substrates, and thapsigargin

Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca(2+)-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1 approximately P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg(2+), conversion of E2 to E1 pattern occurs (even when Ca(2+) is absent) as H(+) dissociates from acidic residues. Mutational analysis demonstrates that the Glu(309) and Glu(771) acidic residues (empty Ca(2+)-binding sites I and II) are required for stabilization of E2. Glu(309) ionization is most important to yield E1. However, a further transition produced by Ca(2+) binding to E1 (i.e. E1.2Ca(2+)) is still needed for catalytic activation. Following ATP utilization, H(+)/Ca(2+) exchange is involved in the transition from the E1 approximately P.2Ca(2+) to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1 approximately P.2Ca(2+) to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca(2+). The E2 state and luminal path closure follow removal of conformational constraint by phosphate.     

The effect of amino acid substitution in the imperfect repeat sequences of α-synuclein on fibrillation

Human α-synuclein is the causative protein of several neurodegenerative diseases, such as Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). The N-terminal half of α-synuclein contains seven imperfect repeat sequences. One of the PD/DLB-causing point mutations, E46K, has been reported in the imperfect repeat sequences of α-synuclein, and is prone to form amyloid fibrils. The presence of seven imperfect repeats in α-synuclein raises the question of whether or not mutations corresponding to E46K in the other imperfect KTKE(Q)GV repeats have similar effects on aggregation and fibrillation, as well as their propensities to form α-helices. To investigate the effect of E(Q)/K mutations in each imperfect repeat sequence, we substituted the amino acid corresponding to E46K in each of the seven repeated sequences with a Lys residue. The mutations in the imperfect KTKE(Q)GV repeat sequences of the N-terminal region were prone to decrease the lag time of fibril formation. In addition, AFM imaging suggested that the Q24K mutant formed twisted fibrils, while the other mutants formed spherical aggregates and short fibrils. These observations indicate that the effect of the mutations on the kinetics of fibril formation and morphology of fibrils varies according to their location.     

Direct insertion of proteins into a living cell using an atomic force microscope with a nanoneedle

We have developed a tool for directly inserting proteins into living cells by using atomic force microscopy (AFM) and an ultrathin needle, termed a nanoneedle. The surface of the nanoneedle was modified with His-tagged proteins using nickel chelating nitrilotriaceticacid (NTA). The fluorescent proteins, DsRed2-His₆ and EGFP-His₆, could be attached to and detached from the surface of the nanoneedle. These results suggest that the Ni-NTA modified nanoneedle can successfully be used for specific delivery of proteins. The nanoneedle modified with DsRed2-His₆ was able to penetrate the surface of a living HeLa cell, as confirmed by laser scanning fluorescence microscopy and monitoring an exerting force on the nanoneedle using AFM. Force curves using the nanoneedle indicated that the needle was able to penetrate at displacement speeds of 0.10–10 μm/s. These results suggest that this technique can be used to directly insert proteins into living cells and is applicable for modulation or regulation of single cell activity.     

Live observation of forespore membrane formation in fission yeast

Sporulation in the fission yeast Schizosaccharomyces pombe is a unique biological process in that the plasma membrane of daughter cells is assembled de novo within the mother cell cytoplasm. A double unit membrane called the forespore membrane (FSM) is constructed dynamically during meiosis. To obtain a dynamic view of FSM formation, we visualized FSM in living cells by using green fluorescent protein fused with Psy1, an FSM-resident protein, together with the nucleus or microtubules. The assembly of FSM initiates in prophase II, and four FSMs in a cell expand in a synchronous manner at the same rate throughout meiosis II. After the meiosis II completes, FSMs continue to expand until closure to form the prespore, a spore precursor. Prespores are initially ellipsoidal, and eventually become spheres. FSM formation was also observed in the sporulation-deficient mutants spo3, spo14, and spo15. In the spo15 mutant, the initiation of FSM formation was completely blocked. In the spo3 mutant, the FSM expanded normally during early meiosis II, but it was severely inhibited during late and postmeiosis, whereas in the spo14 mutant, membrane expansion was more severely inhibited throughout meiosis II. These observations suggest that FSM expansion is composed of two steps, early meiotic FSM expansion and late and post meiotic FSM expansion. Possible regulatory mechanisms of FSM formation in fission yeast are discussed.     

The Schizosaccharomyces pombe cdt2(+) gene,a target of G1-S phase-specific transcription factor complex DSC1, is required for mitotic and premeiotic DNA replication

We have defined five sev genes by genetic analysis of Schizosaccharomyces pombe mutants, which are defective in both proliferation and sporulation. sev1(+)/cdt2(+) was transcribed during the G1-S phase of the mitotic cell cycle, as well as during the premeiotic S phase. The mitotic expression of cdt2(+) was regulated by the MCB-DSC1 system. A mutant of a component of DSC1 affected cdt2(+) expression in vivo, and a cdt2(+) promoter fragment containing MCB motifs bound DSC1 in vitro. Cdt2 protein also accumulated in S phase and localized to the nucleus. cdt2 null mutants grew slowly at 30 degrees and were unable to grow at 19 degrees. These cdt2 mutants were also medially sensitive to hydroxyurea, camptothecin, and 4-nitroquinoline-1-oxide and were synthetically lethal in combination with DNA replication checkpoint mutations. Flow cytometry analysis and pulsed-field gel electrophoresis revealed that S-phase progression was severely retarded in cdt2 mutants, especially at low temperatures. Under sporulation conditions, premeiotic DNA replication was impaired with meiosis I blocked. Furthermore, overexpression of suc22(+), a ribonucleotide reductase gene, fully complemented the sporulation defect of cdt2 mutants and alleviated their growth defect at 19 degrees. These observations suggest that cdt2(+) plays an important role in DNA replication in both the mitotic and the meiotic life cycles of fission yeast.     

Scheduled conversion of replication complex architecture at replication origins of Saccharomyces cerevisiae during the cell cycle

Replication of DNA within Saccharomyces cerevisiae chromosomes is initiated from multiple origins, whose activation follow their own inherent time schedules during the S phase of the cell cycle. It has been demonstrated that a characteristic replicative complex (RC) that includes an origin recognition complex is formed at each origin and shifts between post- and pre-replicative states during the cell cycle. We wanted to determine whether there was an association between this shift in the state of the RC and firing events at replication origins. Time course analyses of RC architecture using UV-footprinting with synchronously growing cells revealed that pre-replicative states at both early and late firing origins appeared simultaneously during late M phase, remained in this state during G(1) phase, and converted to the post-replicative state at various times during S phase. Because the conversion of the origin footprinting profiles and origin firing, as assessed by two-dimensional gel electrophoresis, occurred concomitantly at each origin, then these two events must be closely related. However, conversion of the late firing origin occurred without actual firing. This was observed when the late origin was suppressed in clb5-deficient cells and a replication fork originating from an outside origin replicated the late origin passively. This mechanism ensures that replication at each chromosomal locus occurs only once per cell cycle by shifting existing pre-RCs to the post-RC state, when it is replicated without firing.     

In situ phage screening. A method for identification of subnanogram tissue components in situ

We have established a novel method, in situ phage screening (ISPS), to identify proteins in tissue microstructures. The method is based on the selection of repertoires of phage-displayed antibody fragments with small samples of tissues microdissected using a laser. Using a human muscle frozen section with an area of 4800 microm2 as a model target, we successfully selected monoclonal antibody fragments directed against three major (myosin heavy chain, actin, and tropomyosin-alpha) and one minor (alpha-actinin 2) muscle constituent proteins. These proteins were present in the sample in amounts less than one nanogram, and the antibodies were used to visualize the proteins in situ. This shows that the use of ISPS can obtain monoclonal antibodies for histochemical and biochemical purposes against minute amounts of proteins from microstructures with no requirement for large amounts of samples or biochemical efforts.     

Essential role of ZP molecules in tubal transport of embryos in mice

Our understandings of the molecular and cellular mechanisms underlying tubal transport of embryos are poor. This study describes the essential role of the molecules on the zona pellucida (ZP) in the tubal transport of mouse embryos. The bovine and porcine embryos that were interspecifically transferred to the mouse oviduct were selectively retained in the oviduct and rarely transported to the uterus. Antiserum ZP3-9 against synthetic peptides that are specific for mouse ZP3, significantly interfered with tubal transport of the treated embryos. The treatment of mouse embryos with antiserum ZP2-20 against the synthetic peptides, deduced from the sequences that are conserved in the structure of ZP2 from mouse and human, also inhibited their tubal transport. Among various proteolytic and glycosidic enzymes, treatments with trypsin and beta-glucosidase prior to transfer to the oviduct, significantly interfered with the tubal transport of the enzyme-treated mouse embryos. We hypothesize that species-specific epitopes on the ZP may be recognized by the oviductal cilia and/or the epithelial cells of ducts for tubal transport.