Metabolic O-demethylation does not alter the influence of isoflavones on the biophysical properties of membranes and MRP1-like protein transport activity

2005 ◽  
Vol 433 (2) ◽  
pp. 428-434 ◽  
Author(s):  
Barbara Łania-Pietrzak ◽  
Andrzej B. Hendrich ◽  
Jarosław Żugaj ◽  
Krystyna Michalak
Keyword(s):  
Protein Transport ◽  
Transport Activity ◽  
Biophysical Properties

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

scholarly journals FlhA Undergoes Cyclic Open-close Domain Motions During Flagellar Protein Export in Salmonella

2021 ◽  
Author(s):  
Tohru Minamino ◽  
Yumi Inoue ◽  
Miki Kinoshita ◽  
Akio Kitao ◽  
Keiichi Namba
Keyword(s):  
Closed Form ◽  
Protein Transport ◽  
Building Blocks ◽  
Protein Export ◽  
Side Chain ◽  
Transport Activity ◽  
Flagellar Assembly ◽  
Flagellar Protein ◽  
Domain Motions

Abstract The flagellar type III secretion system (fT3SS) transports flagellar building blocks from the cytoplasm to the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for flagellar chaperones in complex with their cognate substrates and ensures the strict order of protein export for efficient flagellar assembly. FlhAC adopts open and closed conformations, and the chaperones bind to the open form, allowing the fT3SS to transport the substrates to the cell exterior. To clarify the role of the closed form in flagellar protein export, we isolated pseudorevertants from the flhA(G368C/K549C) mutant, in which the closed conformation is stabilized to inhibit the protein transport activity of the fT3SS. Each of M365I, R370S, A446E and P550S substitutions in FlhAC identified in the pseudorevertants affected hydrophobic side-chain interaction networks in the closed FlhAC structure, thereby restoring the protein transport activity to a considerable degree. We propose that a cyclic open-close domain motion of FlhAC is required for rapid and efficient flagellar protein export where a structural transition from the open to the closed form induces the dissociation of empty chaperones from FlhAC.

scholarly journals Identification of a 25-kD protein from yeast cytosol that operates in a prefusion step of vesicular transport between compartments of the Golgi.

1990 ◽  
Vol 110 (4) ◽  
pp. 947-954 ◽  
Author(s):  
B W Wattenberg ◽  
R R Hiebsch ◽  
L W LeCureux ◽  
M P White
Keyword(s):  
Monoclonal Antibodies ◽  
Golgi Apparatus ◽  
Active Component ◽  
Protein Transport ◽  
Transport Activity ◽  
Yeast Protein ◽  
Fusion Event ◽  
Transport Vesicles ◽  
Target Membrane ◽  
Cytosolic Factors

We have identified a 25-kD cytosolic yeast protein that mediates a late, prefusion step in transport of proteins between compartments of the Golgi apparatus. Activity was followed using the previously described cell free assay for protein transport between Golgi compartments as modified to detect late acting cytosolic factors (Wattenberg, B. W., and J. E. Rothman. 1986. J. Biol. Chem. 263:2208-2213). In the reaction mediated by this protein, transport vesicles that have become attached to the target membrane during a preincubation are processed in preparation for fusion. The ultimate fusion event does not require the addition of cytosolic proteins (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:525-536). Although isolated from yeast, this protein has activity when assayed with mammalian membranes. This protein has been enriched over 150-fold from yeast cytosol, albeit not to complete homogeneity. The identity of a 25-kD polypeptide as the active component was confirmed by raising monoclonal antibodies to it. These antibodies were found to specifically inhibit transport activity. Because this is a protein operating in prefusion, it has been abbreviated POP.

Targeting and intracellular trafficking of clinically relevant hTHTR1 mutations in human cell lines

2007 ◽  
Vol 113 (2) ◽  
pp. 93-102 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Jonathan S. Marchant ◽  
Hamid M. Said
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Protein Transport ◽  
Point Mutations ◽  
Sensorineural Deafness ◽  
Normal Growth ◽  
Human Cell Lines ◽  
Transport Activity ◽  
Thiamine Transport ◽  
Mutant Phenotypes

The micronutrient thiamine is required for normal growth and development of human tissues, and is accumulated into cells through the activity of plasma membrane thiamine transporters, e.g. hTHTR1 (human thiamine transporter 1). Recent genetic evidence has linked mutations in hTHTR1 with the manifestation of TRMA (thiamine-responsive megaloblastic anaemia), a condition also associated with diabetes mellitus, sensorineural deafness and retinal disorders. To examine how mutations in hTHTR1 impair thiamine accumulation, we have investigated the targeting and functional properties of several different hTHTR1 mutants in human cell lines derived from epithelia relevant to thiamine absorption or tissues implicated in TRMA pathology. These constructs encompassed two newly identified point mutations (P51L and T158R) and two truncations of hTHTR1 identical with those found in TRMA kindreds (W358X and Δ383fs). Our results reveal a spectrum of mutant phenotypes, underlining that TRMA can result from decreased thiamine transport activity underpinned by changes in hTHTR1 expression levels, cellular targeting and/or protein transport activity.

scholarly journals Lanthanum ions inhibit the mammalian Sec61 complex in its channel dynamics and protein transport activity

FEBS Letters ◽  
2009 ◽  
Vol 583 (14) ◽  
pp. 2359-2364 ◽  
Author(s):  
Frank Erdmann ◽  
Martin Jung ◽  
Susanne Eyrisch ◽  
Sven Lang ◽  
Volkhard Helms ◽  
...  
Keyword(s):  
Protein Transport ◽  
Transport Activity ◽  
Channel Dynamics ◽  
Lanthanum Ions

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 Functional reconstitution of TatB into thylakoidal Tat translocase

2019 ◽  
Author(s):  
Sarah Zinecker ◽  
Mario Jakob ◽  
Ralf Bernd Klösgen
Keyword(s):  
Functional Analysis ◽  
Heterologous Expression ◽  
Protein Transport ◽  
Transmembrane Helix ◽  
Experimental System ◽  
Receptor Complex ◽  
Transport Activity ◽  
E Coli ◽  
Purified Antigen

AbstractWe have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twin-arginine protein transport (Tat1) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analyzed with the authentic Tat substrate pOEC16, reached routinely 20 - 25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutant TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional mutant analyses. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

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.

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