The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy genetika   metabolismus   MeSH
• bakteriální proteiny * metabolismus   MeSH
• nukleotidy metabolismus   MeSH
• proteiny SecA metabolismus   MeSH
• proteiny z Escherichia coli * metabolismus   MeSH
• translokační kanály SEC chemie   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.

• MeSH
• bromkyan metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• cystein metabolismus   MeSH
• dimerizace MeSH
• disulfidy metabolismus   MeSH
• endopeptidasy chemie   metabolismus   ultrastruktura   MeSH
• financování organizované MeSH
• fluorescenční spektrometrie MeSH
• genové produkty gag metabolismus   MeSH
• kinetika MeSH
• Masonův-Pfizerův opičí virus enzymologie   fyziologie   MeSH
• molekulární sekvence - údaje MeSH
• molekulová hmotnost MeSH
• mutantní proteiny chemie   metabolismus   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• posttranslační úpravy proteinů MeSH
• replikace viru fyziologie   MeSH
• retrovirové infekce MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• stabilita enzymů MeSH
• termodynamika MeSH
• virion fyziologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH