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Contents 1 Overview 2 A. Export system 2.1 Introduction 2.2 About signal peptides 2.3 Translocation mechanism

Overview

• Introduction
• A. Export system
• B. Our strategy
• C. Construction

A. Export system

Il faut une phrase qui dit pourquoi vous vous intéressez aux systems d'exportation

Introduction

The Tat (twin-arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues.

The most remarkable characteristic of the Tat pathway is that it apparently functions to transport folded proteins of variable dimensions across the cytoplasmic membrane, a feat that must be achieved without rendering the membrane freely permeable to protons and other ions.

In most cases, the substrates of this pathway are proteins that bind one of a range of cofactors in the cytoplasm and are thus folded before export. Some cofactorless proteins may also be transported by the Tat pathway, probably because they either require cytoplasmic factors for folding or fold too rapidly or tightly for transport by the Sec apparatus.

Recent work has shown that the bacterial Tat system is very closely related to the DeltapH-dependent protein import pathway of the plant chloroplast thylakoid membrane. The bacterial and plant systems do, however, differ in the treatment of precursors before the transport step because, in contrast to well-characterized delta pH-dependent pathway precursors, bacterial Tat substrates has to be co-ordinates with cofactor insertion.

The Sec machinery is composed of a membrane-embedded SecYEG translocation complex + an ATP-hydrolysing SecA protein. Major feature of the Sec mechanism is that proteins are translocated in an extended conformation and are often bound by SecB or other cytoplasmic chaperones to prevent folding before export.

About signal peptides

Sec peptide signal

Sec pathway signal peptides in Gram-negative bacteria are on average 24 amino acids in length and comprise three distinct regions : an N-terminal positively charged region (n-region), a hydrophobic alpha-helical region (h-region) and a c-domain that contains the site of cleavage by signal peptidase.

Tat peptide signal

Tat pathway signal peptides have a similar tripartite organization to Sec signal peptides but exhibit a number of disctinctive features, the most notable of which is a conserved (S/T)-R-R-x-F-L-K sequence motif at the n-region/h-region boundary, in which the consecutive arginine residues are invariant and the other motif residues occur at a frequency of more than 50%.

Tat vs Sec

We observe the high occurrence of proline residues at position -6 to the signal peptidase cleavage site. The c-region of Tat signal peptides also characteristically contains basic amino acids whereas Sec pathway precursors show a bias against positively charged residues in the vicinity of the signal peptidase cleavage site.

Bacterial Tat signal peptides are on average 14 amino acids longer than Sec signal peptides, with most of this additional length being caused by an extended n-region. Further the h-region of the at signal peptides is significantly less hydrophobic than that of Sec signal peptide due to a higher occurrence of the amino acids glycine and threonine and a signifivantly lower abundance of leucine residues.

Targeting to Tat and avoidance of Sec

Increasing the h-region hydrophobicity redirects the protein through the Sec translocon. Thus, co-translational Sec targeting not only overrides Tat targeting information but can also force an otherwise Sec-incompatible wild-type TorA signal sequence into the Sec translocon.

There are many features preventing Tat signals peptides to act as efficiently as Sec targerting signals. It has been shown that the signal peptide c-region basic residues block the Sec-dependent thylakoid import (althought not required for the DeltA pH pathway). The c-region basic residues of bacterial Tat signal peptides also interfere with Sec pathway export. This Sec avoidance characteristic might be connected to the long-recongnized phenomenon that basic residues are poorly tolerated in the vicinity of the signal peptidase cleavage site of Sec pathway precursors. If the h-region hydrophobicity is increased, the export defect caused by basic residues is suppressed. The cytoplasmic membrane potential inhibits insertion of precursors with such basic residues into the Sec translocon.

The twin-arginine consensus has no Sec-avoidance role.

No studies have so far addressed the role of the non-arginine amino-acids of the Tat consensus motif in the transport process.

Translocation mechanism

Sec Mechanism

In the classic Sec loop, the tip of the loop containing the signal peptidase cleavage site exposed at the periplasmic face of the membrane with the two arms of the loop being formed by the inverted signal peptide and approximately the first 20 amino acids of the mature protein. The mature protein arm is subsequently segmentally extruded by SecA insertion cycles.

Tat Mechanism

It has been suggested that, by analogy with protein transport by the Sec pathway and across the endoplasmic reticulum (ER), the Tat system may operate via loop mechanism in which both the N-terminus of the signal peptide and the bulk of the passengers protein remain at the cytoplasmic side of the membrane after the initial translocon-precursor interaction. Weak evidence is available that the N-terminus of the tat signal peptide does indeed remain at the cytoplasmic side of the membrane. The N-terminus of the precursor must remain on the stromal side of the membrane. It remains possible that the mature protein N-terminus does not attain its final conformation until after signal peptide cleavage. If the mature protein is fully folded at the translocon binding step, the signal peptide might pivot around the consensus motif binding site during substrate transport. Alternatively, at the start of transport, when the mature domain is at the cytoplasmic side of the membrane, the signal peptide alone could form a loop that would flex to a fully extended conformation across the membrane by the end of the translocation process.

Folding and cofactors

The cofactor insertion is assisted by dedicated cytoplasmic assembly factors that recognize and bind the precursor. A single signal peptide may even be capable of targeting a three-subunit enzyme though the Tat pathway.

Does the transporter mechanistically require that the substrate be folded ?

No, but maybe the Tat pathway contains elements that check the folding state.

Proofreading would not be a necessity for those Tat substrates that do not bind cofactors, if the reason such proteins are targeted to the Tat pathway is because Sec-dependent export is too slow to prevent the protein forming a Sec-incompatible structure.

A mechanism for checking the cofactor loading will be needed for : tat substrates that binds to cofactors + proteins without cofactors because they require cytosolic folding cofactors. This mechanisme should be able to look to the hydrophobic regions of the preprotein : via chaperonnes or via tat signal peptide.

In all above models, the signal peptide is bifunctionnal : acting both to direct export and to signal the cofactor status. Thus, in addition to the common features of twin arginine signal peptides required for the Tat targeting, signal peptides for the cofactor containing proteins should have distinct structural features allowing specific protein-protein interactions.