Bacterial protein translocation requires only one copy of the SecY complex in vivo

The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between SecY molecules still supported viability of E. coli. These results show that a single SecY molecule is sufficient for protein translocation

Generation of nonidentical compartments in vesicular transport systems

How can organelles communicate by bidirectional vesicle transport and yet maintain different protein compositions? We show by mathematical modeling that a minimal system, in which the basic variables are cytosolic coats for vesicle budding and membrane-bound soluble N-ethyl-maleimide–sensitive factor attachment protein receptors (SNAREs) for vesicle fusion, is sufficient to generate stable, nonidentical compartments. A requirement for establishing and maintaining distinct compartments is that each coat preferentially packages certain SNAREs during vesicle budding. Vesicles fuse preferentially with the compartment that contains the highest concentration of cognate SNAREs, thus further increasing these SNAREs. The stable steady state is the result of a balance between this autocatalytic SNARE accumulation in a compartment and the distribution of SNAREs between compartments by vesicle budding. The resulting nonhomogeneous SNARE distribution generates coat-specific vesicle fluxes that determine the size of compartments. With nonidentical compartments established in this way, the localization and cellular transport of cargo proteins can be explained simply by their affinity for coats

Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1

The toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain. This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol. The polypeptide is initially unfolded by binding to the reduced form of PDI. We show that upon oxidation of the COOH-terminal disulfide bond in PDI by the enzyme Ero1, the A1 chain is released. Both yeast Ero1 and the mammalian Ero1α isoform are active in this reaction. Ero1 has a preference for the PDI–toxin complex. We further show that the complex is transferred to a protein at the lumenal side of the ER membrane, where the unfolded toxin is released from PDI by the action of Ero1. Taken together, our results identify Ero1 as the enzyme mediating the release of unfolded CT from PDI and characterize an additional step in retrotranslocation of the toxin

A “Push and Slide” Mechanism Allows Sequence-Insensitive Translocation of Secretory Proteins by the SecA ATPase

SummaryIn bacteria, most secretory proteins are translocated across the plasma membrane by the interplay of the SecA ATPase and the SecY channel. How SecA moves a broad range of polypeptide substrates is only poorly understood. Here we show that SecA moves polypeptides through the SecY channel by a “push and slide” mechanism. In its ATP-bound state, SecA interacts through a two-helix finger with a subset of amino acids in a substrate, pushing them into the channel. A polypeptide can also passively slide back and forth when SecA is in the predominant ADP-bound state or when SecA encounters a poorly interacting amino acid in its ATP-bound state. SecA performs multiple rounds of ATP hydrolysis before dissociating from SecY. The proposed push and slide mechanism is supported by a mathematical model and explains how SecA allows translocation of a wide range of polypeptides. This mechanism may also apply to hexameric polypeptide-translocating ATPases

A large conformational change of the translocation ATPase SecA

The ATPase SecA mediates the posttranslational translocation of a wide range of polypeptide substrates through the SecY channel in the cytoplasmic membrane of bacteria. We have determined the crystal structure of a monomeric form of Bacillus subtilis SecA at a 2.2-Å resolution. A comparison with the previously determined structures of SecA reveals a nucleotide-independent, large conformational change that opens a deep groove similar to that in other proteins that interact with diverse polypeptides. We propose that the open form of SecA represents an activated state