Gen Matsumoto

SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane

SecA, the preprotein‐driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane‐embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxy‐terminal cytoplasmic domain, that did not allow ATP and preprotein‐dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG ‘channel’ structure in accepting the preprotein‐SecA complex or its opening by the complex. We isolated secA mutations that allele‐specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high‐affinity ATP‐binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.

Microtubules inside the plasma membrane of squid giant axons and their possible physiological function

SummaryThe effects of application of the microtubule-disassembling reagents to squid giant axons upon resting potential, the height of the propagated action potential, and the threshold to evoke action potential were studied using colchicine, podophyllotoxin, vinblastine, griseofulvin, sulfhydryl reagents including NEM, diamide, DTNB and PCMB, and Ca2+ ions. At the same time, the effects of concentrations of K halides and K glutamate on the above physiological properties were studied in comparison within vitro characteristics of microtubule assembly from purified axoplasmic tubulin.It was found that there was good correlation between conditions supporting maintenance of membrane excitability and microtubule assembly. The experiments suggest that associated with the internal surface of the plasma membrane there are microtubules which regulate in part both resting and action potentials.

Restoration of membrane excitability of squid giant axons by reagents activating tyrosine-tubulin ligase

SummaryUsing squid giant axon, an experimental survey was performed on restoration of the membrane excitability which had been partially suppressed. Among reagents examined, a combination of 400mm KF, 50 μm tyrosine, 1mm ATP, 1mm Mg ions and 5 μm cAMP was found to induce the restoration of the excitability to a large extent. Further addition of a small amount of either porcine brain microtubule proteins or the squid axoplasm was found to support complete restoration. The experiments suggest that tubulin-tyrosine ligase contained in the porcine brain microtubule protein fraction or the squid axoplasm maintains the coupling between cytoskeletal structures and the plasma membrane.

Altered accumbens neural response to prediction of reward associated with place in dopamine D2 receptor knockout mice

Midbrain dopaminergic activity seems to be important in forming the prediction of future events such as rewards. The nucleus accumbens (NAc) plays an important role in the integration of reward with motor function, and it receives dense dopamine innervation and extensive limbic and cortical afferents. Here, we examined the specific role of the dopamine D2 receptor (D2R) in mediating associative learning, locomotor activity, and regulating NAc neural responses by using D2R-knockout (KO) mice and their wild-type littermates. D2R-KO mice displayed reduced locomotor activity and slower acquisition of a place-learning task. D2R-KO eliminated the prereward inhibitory response of neurons in the NAc. In contrast, an increased number of neurons in D2R-KO mice displayed place-related activity. These results provide evidence that D2R in the NAc participates in coding for a specific type of neural response to incentive contingencies and partly in spatial learning.

Genetic dissection of SecA: suppressor mutations against the secY205 translocase defect

The driving force for protein translocation across the bacterial plasma membrane is provided by SecA ATPase, which undergoes striking conformational changes characterized by the membrane insertion and deinsertion cycle. This action of SecA requires the membrane‐embedded SecYEG complex. Previously, we have identified a cold‐sensitive secY mutation (secY205), affecting the most carboxy‐terminal cytosolic domain, that did not allow an ATP‐dependent insertion of a SecA‐preprotein complex. Thus, this mutant provides an excellent system for genetic analysis of the SecY–SecA interaction.

A Mutation in secY That Causes Enhanced SecA Insertion and Impaired Late Functions in Protein Translocation

A cold-sensitive secY mutant (secY125) with an amino acid substitution in the first periplasmic domain causes in vivo retardation of protein export. Inverted membrane vesicles prepared from this mutant were as active as the wild-type membrane vesicles in translocation of a minute amount of radioactive preprotein. The mutant membrane also allowed enhanced insertion of SecA, and this SecA insertion was dependent on the SecD and SecF functions. These and other observations suggested that the early events in translocation, such as SecA-dependent insertion of the signal sequence region, is actually enhanced by the SecY125 alteration. In contrast, since the mutant membrane vesicles had decreased capacity to translocate chemical quantity of pro-OmpA and since they were readily inactivated by pretreatment of the vesicles under the conditions in which a pro-OmpA translocation intermediate once accumulated, the late translocation functions appear to be impaired. We conclude that this periplasmic secY mutation causes unbalanced early and late functions in translocation, compromising the translocases ability to catalyze multiple rounds of reactions.

Tyrosinated Tubulin Necessary for Maintenance of Membrane Excitability in Squid Giant Axon

Microtubules are known to play important roles in many cellular functions including motility, mitosis, transport, and the maintenance of cell shape by forming cytoskeletons. Nerve tissues contain a large amount of tubulin and microtubules, and evidence has been accumulated in support of the view that neuronal microtubules are directly involved in axonal transport (Abe et al., 1973; James et al., 1970).