scholarly journals Component Specificity for the Thylakoidal Sec and Delta Ph–Dependent Protein Transport Pathways

1999 ◽  
Vol 146 (1) ◽  
pp. 45-56 ◽  
Author(s):  
Hiroki Mori ◽  
Elizabeth J. Summer ◽  
Xianyue Ma ◽  
Kenneth Cline
Keyword(s):  
Protein Transport ◽  
Signal Recognition Particle ◽  
Thylakoid Membranes ◽  
Transport Pathways ◽  
Sec Pathway ◽  
Evolutionarily Conserved ◽  
In Series ◽  
Dependent Protein ◽  
Membrane Components ◽  
Substrate Proteins

Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in ∼10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.

scholarly journals Stoichiometry for binding and transport by the twin arginine translocation system

2012 ◽  
Vol 197 (4) ◽  
pp. 523-534 ◽  
Author(s):  
Jose M. Celedon ◽  
Kenneth Cline
Keyword(s):  
Kinetic Analysis ◽  
Protein Transport ◽  
Thylakoid Membranes ◽  
Oxygen Evolving Complex ◽  
Receptor Complex ◽  
Twin Arginine Translocation ◽  
Precursor Proteins ◽  
Folded Proteins ◽  
Membrane Components ◽  
Oxygen Evolving

Twin arginine translocation (Tat) systems transport large folded proteins across sealed membranes. Tat systems accomplish this feat with three membrane components organized in two complexes. In thylakoid membranes, cpTatC and Hcf106 comprise a large receptor complex containing an estimated eight cpTatC-Hcf106 pairs. Protein transport occurs when Tha4 joins the receptor complex as an oligomer of uncertain size that is thought to form the protein-conducting structure. Here, binding analyses with intact membranes or purified complexes indicate that each receptor complex could bind eight precursor proteins. Kinetic analysis of translocation showed that each precursor-bound site was independently functional for transport, and, with sufficient Tha4, all sites were concurrently active for transport. Tha4 titration determined that ∼26 Tha4 protomers were required for transport of each OE17 (oxygen-evolving complex subunit of 17 kD) precursor protein. Our results suggest that, when fully saturated with precursor proteins and Tha4, the Tat translocase is an ∼2.2-megadalton complex that can individually transport eight precursor proteins or cooperatively transport multimeric precursors.

scholarly journals Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Iain D. Hay ◽  
Matthew J. Belousoff ◽  
Trevor Lithgow
Keyword(s):  
Outer Membrane ◽  
Protein Transport ◽  
Substrate Uptake ◽  
Inner Membrane ◽  
Protein Substrates ◽  
Folded Proteins ◽  
Membrane Components ◽  
Substrate Proteins ◽  
Vexing Question

ABSTRACT Sophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner membrane and a secretin complex in the outer membrane. The engagement of these two megadalton-sized complexes is required in order to secrete toxins, effectors, and hydrolytic enzymes. Pseudomonas aeruginosa has at least two T2SSs, with the ancestral nanomachine having a secretin complex composed of XcpQ. Until now, no high-resolution structural information was available to distinguish the features of this Pseudomonas-type secretin, which varies greatly in sequence from the well-characterized Klebsiella-type and Vibrio-type secretins. We have purified the ~1-MDa secretin complex and analyzed it by cryo-electron microscopy. Structural comparisons with the Klebsiella-type secretin complex revealed a striking structural homology despite the differences in their sequence characteristics. At 3.6-Å resolution, the secretin complex was found to have 15-fold symmetry throughout the membrane-embedded region and through most of the domains in the periplasm. However, the N1 domain and N0 domain were not well ordered into this 15-fold symmetry. We suggest a model wherein this disordering of the subunit symmetry for the periplasmic N domains provides a means to engage with the 6-fold symmetry in the inner membrane platform, with a metastable engagement that can be disrupted by substrate proteins binding to the region between XcpP, in the assembly platform, and the XcpQ secretin. IMPORTANCE How the outer membrane and inner membrane components of the T2SS engage each other and yet can allow for substrate uptake into the secretin chamber has challenged the protein transport field for some time. This vexing question is of significance because the T2SS collects folded protein substrates in the periplasm for transport out of the bacterium and yet must discriminate these few substrate proteins from all the other hundred or so folded proteins in the periplasm. The structural analysis here supports a model wherein substrates must compete against a metastable interaction between XcpP in the assembly platform and the XcpQ secretin, wherein only structurally encoded features in the T2SS substrates compete well enough to disrupt XcpQ-XcpP for entry into the XcpQ chamber, for secretion across the outer membrane. IMPORTANCE How the outer membrane and inner membrane components of the T2SS engage each other and yet can allow for substrate uptake into the secretin chamber has challenged the protein transport field for some time. This vexing question is of significance because the T2SS collects folded protein substrates in the periplasm for transport out of the bacterium and yet must discriminate these few substrate proteins from all the other hundred or so folded proteins in the periplasm. The structural analysis here supports a model wherein substrates must compete against a metastable interaction between XcpP in the assembly platform and the XcpQ secretin, wherein only structurally encoded features in the T2SS substrates compete well enough to disrupt XcpQ-XcpP for entry into the XcpQ chamber, for secretion across the outer membrane.

scholarly journals Proteomics Screen Identifies Class I Rab11 Family Interacting Proteins as Key Regulators of Cytokinesis

2016 ◽  
Vol 37 (3) ◽  
Author(s):  
Carl Laflamme ◽  
Jacob A. Galan ◽  
Khaled Ben El Kadhi ◽  
Antoine Méant ◽  
Carlos Zeledon ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Trafficking ◽  
Protein Transport ◽  
Effector Proteins ◽  
Class I ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Content Type ◽  
Evolutionarily Conserved ◽  
Dependent Protein

ABSTRACTThe 14-3-3 protein family orchestrates a complex network of molecular interactions that regulates various biological processes. Owing to their role in regulating the cell cycle and protein trafficking, 14-3-3 proteins are prevalent in human diseases such as cancer, diabetes, and neurodegeneration. 14-3-3 proteins are expressed in all eukaryotic cells, suggesting that they mediate their biological functions through evolutionarily conserved protein interactions. To identify these core 14-3-3 client proteins, we used an affinity-based proteomics approach to characterize and compare the human andDrosophila14-3-3 interactomes. Using this approach, we identified a group of Rab11 effector proteins, termed class I Rab11 family interacting proteins (Rab11-FIPs), or Rip11 inDrosophila. We found that 14-3-3 binds to Rip11 in a phospho-dependent manner to ensure its proper subcellular distribution during cell division. Our results indicate that Rip11 plays an essential role in the regulation of cytokinesis and that this function requires its association with 14-3-3 but not with Rab11. Together, our results suggest an evolutionarily conserved role for 14-3-3 in controlling Rip11-dependent protein transport during cytokinesis.

Protein translocation across the inner membrane of Gram-negative bacteria: the Sec and Tat dependent protein transport pathways

2013 ◽  
Vol 164 (6) ◽  
pp. 505-534 ◽  
Author(s):  
Renuka Kudva ◽  
Kärt Denks ◽  
Patrick Kuhn ◽  
Andreas Vogt ◽  
Matthias Müller ◽  
...  
Keyword(s):  
Protein Transport ◽  
Protein Translocation ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Transport Pathways ◽  
Gram Negative ◽  
Dependent Protein

scholarly journals Regulation of Cardiac PKA Signaling by cAMP and Oxidants

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 663
Author(s):  
Friederike Cuello ◽  
Friedrich W. Herberg ◽  
Konstantina Stathopoulou ◽  
Philipp Henning ◽  
Simon Diering
Keyword(s):  
Contractile Function ◽  
Cardiac Contractility ◽  
Cellular Protein ◽  
Cardiac Contractile Function ◽  
Cardiac Contractile ◽  
Main Driver ◽  
Dependent Protein ◽  
Relevant Regulation ◽  
Protein Dysfunction ◽  
Substrate Proteins

Pathologies, such as cancer, inflammatory and cardiac diseases are commonly associated with long-term increased production and release of reactive oxygen species referred to as oxidative stress. Thereby, protein oxidation conveys protein dysfunction and contributes to disease progression. Importantly, trials to scavenge oxidants by systemic antioxidant therapy failed. This observation supports the notion that oxidants are indispensable physiological signaling molecules that induce oxidative post-translational modifications in target proteins. In cardiac myocytes, the main driver of cardiac contractility is the activation of the β-adrenoceptor-signaling cascade leading to increased cellular cAMP production and activation of its main effector, the cAMP-dependent protein kinase (PKA). PKA-mediated phosphorylation of substrate proteins that are involved in excitation-contraction coupling are responsible for the observed positive inotropic and lusitropic effects. PKA-actions are counteracted by cellular protein phosphatases (PP) that dephosphorylate substrate proteins and thus allow the termination of PKA-signaling. Both, kinase and phosphatase are redox-sensitive and susceptible to oxidation on critical cysteine residues. Thereby, oxidation of the regulatory PKA and PP subunits is considered to regulate subcellular kinase and phosphatase localization, while intradisulfide formation of the catalytic subunits negatively impacts on catalytic activity with direct consequences on substrate (de)phosphorylation and cardiac contractile function. This review article attempts to incorporate the current perception of the functionally relevant regulation of cardiac contractility by classical cAMP-dependent signaling with the contribution of oxidant modification.

scholarly journals Genetic Analysis of Pathway Specificity during Posttranslational Protein Translocation across the Escherichia coli Plasma Membrane

2003 ◽  
Vol 185 (9) ◽  
pp. 2811-2819 ◽  
Author(s):  
Natascha Blaudeck ◽  
Peter Kreutzenbeck ◽  
Roland Freudl ◽  
Georg A. Sprenger
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Protein Translocation ◽  
Alternative Possibilities ◽  
Amino Acid Residues ◽  
Sec Pathway ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Arginine Residues ◽  
Dependent Protein

ABSTRACT In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the “Sec avoidance signal,” the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

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