In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis

The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers

Characterization of the annular lipid shell of the Sec translocon

The bacterial Sec translocase in its minimal form consists of a membrane-embedded protein-conducting pore SecYEG that interacts with the motor protein SecA to mediate the translocation of secretory proteins. In addition, the SecYEG translocon interacts with the accessory SecDFyajC membrane complex and the membrane protein insertase YidC. To examine the composition of the native lipid environment in the vicinity of the SecYEG complex and its impact on translocation activity, styrene-maleic acid lipid particles (SMALPs) were used to extract SecYEG with its lipid environment directly from native Escherichia coli membranes without the use of detergents. This allowed the co-extraction of SecYEG in complex with SecA, but not with SecDFyajC or YidC. Lipid analysis of the SecYEG-SMALPs revealed an enrichment of negatively charged lipids in the vicinity of SecYEG, which in detergent assisted reconstitution of the Sec translocase are crucial for the translocation activity. Such lipid enrichment was not found with separately extracted SecDFyajC or YidC, which demonstrates a specific interaction between SecYEG and negatively charged lipids

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH

Functional studies of the Sec translocase: From in vitro to in vivo

In bacteria, more than one-third of the proteins synthesized in the cell accomplish their function outside the cytoplasm. For this reason, cells contain specialized transport systems that are responsible for the translocation or insertion of proteins across or into the cytoplasmic membrane. The majority of these proteins are transported via the Sec system that is universally conserved in all kingdoms of life. This system contains two main components, a protein-conducting channel across the membrane, SecYEG, and a molecular motor SecA that drives unfolded proteins through the channel. Some pathogenic bacteria contain a second SecA protein, SecA2 that is involved in the secretion of virulence factors. Our studies show that SecA1 and SecA2 interact, providing an explanation why some proteins require both SecA proteins for secretion. By using a single molecule approach in living cells, we show that the Sec system is randomly distributed across the cytoplasmic membrane as two distinct species that exhibit different membrane diffusion characteristics, likely an active and an idle complex. The work provides a deeper understanding of the mechanism of protein translocation and the molecular basis of bacterial virulence

The Canonical and Accessory Sec System of Gram-positive Bacteria

The Sec system is present in all bacteria and responsible for the translocation of the majority of proteins across the cytoplasmic membrane. The system consists of two principal components: the ATPase motor protein, SecA, and the protein-conducting channel, SecYEG. In addition to this canonical Sec system, several Gram-positive bacteria also possess a so-called accessory Sec system. This is a specialized translocation system that is responsible for the export of a subset of secretory proteins, including virulence factors. The accessory Sec system consists of a second SecA paralog, termed SecA2, with or without a second SecY paralog, termed SecY2. In some bacteria, the accessory Sec system is dependent on the canonical Sec system for functionality, while in other bacteria, they can function independently. In this review, we provide an overview of the current knowledge of the canonical and accessory Sec system of Gram-positive bacteria with a focus on the primary component of the Sec translocase, SecA and SecYEG.</p

The Canonical and Accessory Sec System of Gram-positive Bacteria

The Sec system is present in all bacteria and responsible for the translocation of the majority of proteins across the cytoplasmic membrane. The system consists of two principal components: the ATPase motor protein, SecA, and the protein-conducting channel, SecYEG. In addition to this canonical Sec system, several Gram-positive bacteria also possess a so-called accessory Sec system. This is a specialized translocation system that is responsible for the export of a subset of secretory proteins, including virulence factors. The accessory Sec system consists of a second SecA paralog, termed SecA2, with or without a second SecY paralog, termed SecY2. In some bacteria, the accessory Sec system is dependent on the canonical Sec system for functionality, while in other bacteria, they can function independently. In this review, we provide an overview of the current knowledge of the canonical and accessory Sec system of Gram-positive bacteria with a focus on the primary component of the Sec translocase, SecA and SecYEG

Characterization of the annular lipid shell of the Sec translocon

The bacterial Sec translocase in its minimal form consists of a membrane-embedded protein-conducting pore SecYEG that interacts with the motor protein SecA to mediate the translocation of secretory proteins. In addition, the SecYEG translocon interacts with the accessory SecDFyajC membrane complex and the membrane protein insertase YidC. To examine the composition of the native lipid environment in the vicinity of the SecYEG complex and its impact on translocation activity, styrene-maleic acid lipid particles (SMALPs) were used to extract SecYEG with its lipid environment directly from native Escherichia coli membranes without the use of detergents. This allowed the co-extraction of SecYEG in complex with SecA, but not with SecDFyajC or YidC. Lipid analysis of the SecYEG-SMALPs revealed an enrichment of negatively charged lipids in the vicinity of SecYEG, which in detergent assisted reconstitution of the Sec translocase are crucial for the translocation activity. Such lipid enrichment was not found with separately extracted SecDFyajC or YidC, which demonstrates a specific interaction between SecYEG and negatively charged lipids.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Overexpression, purification, and fluorescent labeling of <i>M</i>. <i>tuberculosis</i> SecA1 and SecA2.

<p>A) Overexpression of <i>M</i>. <i>tuberculosis</i> SecA1 (lane 2) and SecA2 (lane 4) in <i>E</i>. <i>coli</i> BL21(λDE3) with a molecular mass of 105 and 85 kDa, respectively. Molecular masses of protein standard are indicated on the left. The empty vector, pACYCDuet-1 and pET15b, are shown as controls (lane 1 and 3). B) Coomassie-stained SDS-PAGE of purified <i>M</i>. <i>tuberculosis</i> SecA1 (lane 1) and SecA2 (lane 2). C) Visualization of fluorescently labeled SecA1 and SecA2 in SDS-PAGE by fluorescence imaging. SecA1 and SecA2 were labeled with the fluorescent probe Cy5 (lane 1 and 2) or AF488 (lane 3 and 4).</p

MST analysis of SecA1 and SecA2 homodimerization.

<p>A) Scheme of the MST experiments and B) diffusion traces observed in MST. The mobility of molecules in a temperature gradient is followed by the fluorescence intensity in a central spot. When the IR-Laser is turned on, the initial fluorescence (1) drops due to the thermophoretic movement of fluorescently labeled proteins out of the heated spot (2). When the IR-Laser is turned off, back-diffusion of the fluorescently labeled proteins is observed which is driven by mass diffusion and depends on the hydration shell of the proteins (3). Dimers diffuse slower than monomers. SecA1 (C) and SecA2 (D) dimerization measured by MST. Unlabeled protein (1 nM to 10 μM) was titrated into a fixed concentration of labeled protein (25 nM). The thermophoretic signal is plotted as a function of the protein concentration resulting in a dimerization curve. The curves were fitted using the Hill-equation and apparent K_d values were determined. Error bars represent the standard error of 3 measurements. The apparent K_d for SecA1 and SecA2 dimerization at low salt concentrations were 65 ± 2.5 nM and 161 ± 6.2 nM, respectively. The measurement of samples at high salt concentrations showed no binding.</p