scholarly journals Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

2020 ◽  
Author(s):  
William J. Allen ◽  
Daniel W. Watkins ◽  
Mark S. Dillingham ◽  
Ian Collinson
Keyword(s):  
Protein Transport ◽  
Atp Hydrolysis ◽  
Kinetic Modelling ◽  
Membrane Transporters ◽  
Transport Activity ◽  
Natural Tags ◽  
Indirect Coupling ◽  
Sec System ◽  
Rate Limiting ◽  
Best Fit

AbstractThe universally conserved Sec system is the primary method cells utilise to transport proteins across membranes. Until recently, measuring the activity – a prerequisite for understanding how biological systems works – has been limited to discontinuous protein transport assays with poor time resolution, or used as reporters large, non-natural tags that interfere with the process. The development of an assay based on a split super-bright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute post-translational transport in bacteria. Under the conditions deployed, transport of the model pre-protein substrate proSpy occurs at 200 amino acids per minute with the data best fit by a series of large, ∼30 amino acid, steps each coupled to many (100s) ATP hydrolysis events. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modelling suggests that SecA-driven transport activity is facilitated by the substrate (polypeptide) concentration gradient – in keeping with classical membrane transporters. Furthermore, the features we describe are consistent with a non-deterministic motor mechanism, such as a Brownian ratchet.

scholarly journals Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

2020 ◽  
Vol 117 (50) ◽  
pp. 31808-31816
Author(s):  
William J. Allen ◽  
Daniel W. Watkins ◽  
Mark S. Dillingham ◽  
Ian Collinson
Keyword(s):  
Amino Acids ◽  
Kinetic Modeling ◽  
Time Resolution ◽  
Protein Transport ◽  
Atp Hydrolysis ◽  
Transport Proteins ◽  
Primary Method ◽  
Transport Activity ◽  
Sec System ◽  
Rate Limiting

The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity—a prerequisite for understanding how biological systems work—has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

scholarly journals Bacterial Sec Protein Transport Is Rate-limited by Precursor Length: A Single Turnover Study

2009 ◽  
Vol 20 (19) ◽  
pp. 4256-4266 ◽  
Author(s):  
Fu-Cheng Liang ◽  
Umesh K. Bageshwar ◽  
Siegfried M. Musser
Keyword(s):  
Protein Transport ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Precursor Proteins ◽  
Inverted Membrane Vesicles ◽  
Rate Limiting

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.

scholarly journals Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex

2001 ◽  
Vol 155 (3) ◽  
pp. 369-380 ◽  
Author(s):  
Hein Sprong ◽  
Sophie Degroote ◽  
Tijs Claessens ◽  
Judith van Drunen ◽  
Viola Oorschot ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Direct Pathway ◽  
Mouse Melanoma Cell ◽  
Multicellular Organisms ◽  
Rate Limiting ◽  
Tyrosinase Related Protein ◽  
Mouse Melanoma Cell Line

A;lthough glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.

Surface Separation Equilibria and Dynamics of Cationic Dye Loaded onto Citric Acid and Sodium Hydroxide Treated Eggshells

2018 ◽  
Vol 17 (1) ◽  
Author(s):  
Elisha A. Adeniji ◽  
Temitope O. Abodunrin ◽  
Temitope A. Ogunnupebi ◽  
Babatunde A. Koiki ◽  
Abimbola M. Olatunde ◽  
...  
Keyword(s):  
Citric Acid ◽  
Diffusion Mechanism ◽  
Experiment Data ◽  
Surface Separation ◽  
Rate Limiting Step ◽  
Monolayer Adsorption ◽  
Delta G ◽  
Rate Limiting ◽  
Best Fit ◽  
Adsorption Systems

AbstractThis research enthusiastically highlights the bio-adsorption of methylene blue (MB) by local, poultry, NaOH and citric acid modified ubiquitous eggshell (LES, NLES, CLES, PES, NPES and CPES) adsorbents. The microstructures of these adsorbents indicated that they had some surface functional moieties that were responsible for the adsorption of MB. The Langmuir isotherm and PSO model best fit the experiment data. The largest Langmuir monolayer adsorption capacity${q_{max}}$, was 242.47 mg/g, with the largest MB initial concentration of 400 mg/L. This was a clear indication and a confirmation that MB adsorption by the powdered eggshells was chemisorptive. Moreover, the values of$F$, the thickness of the boundary layer/film were$\gt 0$, showing that the rate limiting step for the adsorption process was controlled by more than one diffusion mechanism. The values of$\Delta {G^\circ }$for the adsorption of MB by the adsorbents indicated that the adsorption reactions were all non-feasible and non-spontaneous. The values for$\Delta {S^\circ }$(J/K/mol) for LES, NLES and CPES for the uptake of MB showed decrease in the chaos or degree of randomness of the adsorption reactions, and the reverse was the case for PES, NPES and CLES for the uptake of MB, which showed increase in the chaos or degree of randomness of the adsorption. The adsorption of MB by LES, NLES and CPES gave$\Delta {H^\circ }$(kJ/mol) values which were indicative of endothermic nature of the adsorption systems, and the reverse was the case for the uptake of MB by PES, NPES and CLES, which was indicative of the exothermic nature of the adsorption systems.

Exercise training increases the number of glucose transporters in rat adipose cells

1989 ◽  
Vol 257 (4) ◽  
pp. E520-E530
Author(s):  
M. F. Hirshman ◽  
L. J. Wardzala ◽  
L. J. Goodyear ◽  
S. P. Fuller ◽  
E. D. Horton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Glucose Transporters ◽  
Membrane Transporters ◽  
Control Group ◽  
Transport Activity ◽  
Insulin Stimulation ◽  
Adipose Cell ◽  
Adipose Cell Size ◽  
Adipose Cells

We studied the mechanism for the increase in glucose transport activity that occurs in adipose cells of exercise-trained rats. Glucose transport activity, glucose metabolism, and the subcellular distribution of glucose transporters were measured in adipose cells from rats raised in wheel cages for 6 wk (mean total exercise 350 km/rat), age-matched sedentary controls, and young sedentary controls matched for adipose cell size. Basal rates of glucose transport and metabolism were greater in cells from exercise-trained rats compared with young controls, and insulin-stimulated rates were greater in the exercise-trained rats compared with both age-matched and young controls. The numbers of plasma membrane glucose transporters were not different among groups in the basal state; however, with insulin stimulation, cells from exercise-trained animals had significantly more plasma membrane transporters than young controls or age-matched controls. Exercise-trained rats also had more low-density microsomal transporters than control rats in the basal state. When the total number of glucose transporters/cell was calculated, the exercise-trained rats had 42% more transporters than did either control group. These studies demonstrate that the increased glucose transport and metabolism observed in insulin-stimulated adipose cells from exercise-trained rats is due, primarily, to an increase in the number of plasma membrane glucose transporters translocated from an enlarged intracellular pool.

scholarly journals Kinetic modelling of hydrogen transfer deoxygenation of a prototypical fatty acid over a bimetallic Pd60Cu40 catalyst: an investigation of the surface reaction mechanism and rate limiting step

2020 ◽  
Vol 5 (9) ◽  
pp. 1682-1693
Author(s):  
Kin Wai Cheah ◽  
Suzana Yusup ◽  
Martin J. Taylor ◽  
Bing Shen How ◽  
Amin Osatiashtiani ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Reaction Mechanism ◽  
Hydrogen Transfer ◽  
Surface Reaction ◽  
Kinetic Modelling ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cu Catalyst ◽  
P Application ◽  
Rate Limiting

Application of tetralin as a source of hydrogen for catalytic conversion of oleic acid to diesel-like hydrocarbons using a bimetallic Pd–Cu catalyst.

scholarly journals Substrate Proteins Take Shape at an Improved Bacterial Translocon

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Donald Oliver
Keyword(s):  
Protein Transport ◽  
Protein Translocation ◽  
Membrane Vesicles ◽  
Transport Models ◽  
Content Type ◽  
Rate Limiting ◽  
Substrate Proteins

ABSTRACTCharacterization of Sec-dependent bacterial protein transport has often relied on anin vitroprotein translocation system comprised in part ofEscherichia coliinverted inner membrane vesicles or, more recently, purified SecYEG translocons reconstituted into liposomes using mostly a single substrate (proOmpA). A paper published in this issue (P. Bariya and L. Randall, J Bacteriol 201:e00493-18, 2019, https://doi.org/10.1128/JB.00493-18) finds that inclusion of SecA protein during SecYEG proteoliposome reconstitution dramatically improves the number of active translocons. This experimentally useful and intriguing result that may arise from SecA membrane integration properties is discussed here. Furthermore, determination of the rate-limiting transport step for nine different substrates implicates the mature region distal to the signal peptide in the observed rate constant differences, indicating that more nuanced transport models that respond to differences in protein sequence and structure are needed.

Titration of protein transport activity by incremental changes in signal peptide hydrophobicity

Biochemistry ◽  
1993 ◽  
Vol 32 (5) ◽  
pp. 1251-1256 ◽  
Author(s):  
Suzanne K. Doud ◽  
Margaret M. Chou ◽  
Debra A. Kendall
Keyword(s):  
Signal Peptide ◽  
Protein Transport ◽  
Transport Activity ◽  
Peptide Hydrophobicity
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