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

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.

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Signal peptide-chaperone interactions on the twin-arginine protein transport pathway

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0500737102 ◽

2005 ◽

Vol 102 (24) ◽

pp. 8460-8465 ◽

Cited By ~ 68

Author(s):

K. Hatzixanthis ◽

T. A. Clarke ◽

A. Oubrie ◽

D. J. Richardson ◽

R. J. Turner ◽

...

Keyword(s):

Signal Peptide ◽

Protein Transport ◽

Transport Pathway ◽

Chaperone Interactions

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

The Journal of Cell Biology ◽

10.1083/jcb.110.4.947 ◽

1990 ◽

Vol 110 (4) ◽

pp. 947-954 ◽

Cited By ~ 12

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.

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

Clinical Science ◽

10.1042/cs20060331 ◽

2007 ◽

Vol 113 (2) ◽

pp. 93-102 ◽

Cited By ~ 9

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.

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Signal Peptide Hydrophobicity Modulates Interaction with the Twin-Arginine Translocase

mBio ◽

10.1128/mbio.00909-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 12

Author(s):

Qi Huang ◽

Tracy Palmer

Keyword(s):

Signal Peptide ◽

Secretory Pathway ◽

Recognition Site ◽

Signal Peptides ◽

Tat Pathway ◽

Folded Proteins ◽

Twin Arginine Translocase ◽

Critical Requirement ◽

Highly Hydrophobic ◽

Peptide Hydrophobicity

ABSTRACT The general secretory pathway (Sec) and twin-arginine translocase (Tat) operate in parallel to export proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Substrates are targeted to their respective machineries by N-terminal signal peptides that share a tripartite organization; however, Tat signal peptides harbor a conserved and almost invariant arginine pair that is critical for efficient targeting to the Tat machinery. Tat signal peptides interact with a membrane-bound receptor complex comprised of TatB and TatC components, with TatC containing the twin-arginine recognition site. Here, we isolated suppressors in the signal peptide of the Tat substrate, SufI, that restored Tat transport in the presence of inactivating substitutions in the TatC twin-arginine binding site. These suppressors increased signal peptide hydrophobicity, and copurification experiments indicated that they restored binding to the variant TatBC complex. The hydrophobic suppressors could also act in cis to suppress substitutions at the signal peptide twin-arginine motif that normally prevent targeting to the Tat pathway. Highly hydrophobic variants of the SufI signal peptide containing four leucine substitutions retained the ability to interact with the Tat system. The hydrophobic signal peptides of two Sec substrates, DsbA and OmpA, containing twin lysine residues, were shown to mediate export by the Tat pathway and to copurify with TatBC. These findings indicate that there is unprecedented overlap between Sec and Tat signal peptides and that neither the signal peptide twin-arginine motif nor the TatC twin-arginine recognition site is an essential mechanistic feature for operation of the Tat pathway. IMPORTANCE Protein export is an essential process in all prokaryotes. The Sec and Tat export pathways operate in parallel, with the Sec machinery transporting unstructured precursors and the Tat pathway transporting folded proteins. Proteins are targeted to the Tat pathway by N-terminal signal peptides that contain an almost invariant twin-arginine motif. Here, we make the surprising discovery that the twin arginines are not essential for recognition of substrates by the Tat machinery and that this requirement can be bypassed by increasing the signal peptide hydrophobicity. We further show that signal peptides of bona fide Sec substrates can also mediate transport by the Tat pathway. Our findings suggest that key features of the Tat targeting mechanism have evolved to prevent mistargeting of substrates to the Sec pathway rather than being a critical requirement for function of the Tat pathway.

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Annotating eukaryotic and toxin-specific signal peptides using Razor

10.1101/2020.11.30.405613 ◽

2020 ◽

Author(s):

Bikash K. Bhandari ◽

Paul P. Gardner ◽

Chun Shen Lim

Keyword(s):

Signal Peptide ◽

Web Application ◽

Protein Transport ◽

Protein Translocation ◽

Recombinant Protein Expression ◽

Disease Diagnosis ◽

Signal Peptides ◽

Specific Signal ◽

Link Type ◽

Command Line Tool

ABSTRACTMotivationSignal peptides are responsible for protein transport and secretion and are ubiquitous to all forms of life. The annotation of signal peptides is important for understanding protein translocation and toxin secretion, optimising recombinant protein expression, as well as for disease diagnosis and metagenomics.ResultsHere we explore the features of these signal sequences across eukaryotes. We find that different kingdoms have their characteristic distributions of signal peptide residues. Additionally, the signal peptides of secretory toxins have common features across kingdoms. We leverage these subtleties to build Razor, a simple yet powerful tool for annotating signal peptides, which additionally predicts toxin- and fungal-specific signal peptides based on the first 23 N-terminal residues. Finally, we demonstrate the usability of Razor by scanning all reviewed sequences from UniProt. Indeed, Razor is able to identify toxins using their signal peptide sequences only. Strikingly, we discover that many defensive proteins across kingdoms harbour a toxin-like signal peptide; some of these defensive proteins have emerged through convergent evolution, e.g. defensin and defensin-like protein families, and phospholipase families.Availability and implementationRazor is available as a web application (https://tisigner.com/razor) and a command-line tool (https://github.com/Gardner-BinfLab/Razor).

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