Chara corallina class XI myosin is by far the fastest molecular motor. To investigate the molecular mechanism of this fast movement, we performed a kinetic analysis of a recombinant motor domain of Chara myosin. We estimated the time spent in the strongly bound state with actin by measuring rate constants of ADP dissociation from actin⅐motor domain complex and ATP-induced dissociation of the motor domain from actin. The rate constant of ADP dissociation from acto-motor domain was >2800 s ؊1 , and the rate constant of ATP-induced dissociation of the motor domain from actin at physiological ATP concentration was 2200 s ؊1 . From these data, the time spent in the strongly bound state with actin was estimated to be <0.82 ms. This value is the shortest among known values for various myosins and yields the duty ratio of <0.3 with a V max value of the actin-activated ATPase activity of 390 s ؊1 . The addition of the long neck domain of myosin Va to the Chara motor domain largely increased the velocity of the motility without increasing the ATP hydrolysis cycle rate, consistent with the swinging lever model. In addition, this study reveals some striking kinetic features of Chara myosin that are suited for the fast movement: a dramatic acceleration of ADP release by actin (1000-fold) and extremely fast ATP binding rate.Myosin is an actin-based motor protein that converts chemical energy liberated by the ATP hydrolysis into directed movement of actin filaments. Phylogenetic analysis of myosin sequences revealed that there are at least 24 classes of myosin (1). Motor functions, such as motility and ATP hydrolysis activity, vary significantly among these classes of myosin. Among those known so far, myosin from alga Chara corallina is the fastest, moving actin filaments at 40 -60 m/s in the in vitro motility assay (2-4). This velocity is ϳ10 times faster than that of the fast skeletal muscle myosin (5). We have been interested in the mechanism how this plant myosin can move so fast. To understand the molecular mechanism of the fast movement,

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Neuronal activity has an impact on β cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-β peptide (Aβ). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of β cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1–containing microdomains through X11–Munc18, which inhibits the APP–BACE1 interaction and β cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Aβ overproduction, promotes the switching of APP microdomain association as well as β cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

BackgroundGenetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.Methodology/Principal FindingsHere we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.Conclusions and SignificanceThe multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.

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