(B) In the presence of stabilizing mutations or exogenous factors such as chemical or protein chaperones that bind to the test protein and prevent aggregation, chimeras such as ssTorA-A42-Bla remain soluble and are efficiently exported to the periplasm where Bla hydrolyzes Amp

(B) In the presence of stabilizing mutations or exogenous factors such as chemical or protein chaperones that bind to the test protein and prevent aggregation, chimeras such as ssTorA-A42-Bla remain soluble and are efficiently exported to the periplasm where Bla hydrolyzes Amp. folding quality control, high-throughput screening, protein misfolding disorders, protein secretion, -synuclein Maintenance of proteome integrity (proteostasis) is essential for cellular and organismal survival, and represents a major challenge across all kingdoms of life. Proteostasis entails highly integrated cellular networks that generate and safeguard the protein fold. 1 Even in simple organisms, such as proteome is usually localized partially Phellodendrine chloride or completely outside of the cytosol,3 which requires insertion into or passage across at least one hydrophobic lipid bilayer membrane. In many instances, the process of membrane translocation is dependent on proper structural integrity of the protein to be transported. For example, the translocase of the Sec protein export pathway provides an aqueous Phellodendrine chloride channel that is approximately the same width as a polypeptide chain (estimated as 15C20 ? on the basis of the crystal structure).4 Given such a narrow pore, the translocase can tolerate polypeptides that form an -helix but not tertiary structure; hence, Sec substrates must be transported in an unfolded state.4,5 The task of Phellodendrine chloride preventing premature folding of Sec substrates prior to translocation is performed in part by a chaperone network, which in consists of GroEL, SecB and trigger factor.6,7 These chaperones bind Sec substrates during or just after translation and provide an important QC layer to the Sec pathway by effectively maintaining the polypeptide chains in Rabbit polyclonal to PHF13 a conformation suitable for transport and preventing illicit interactions between these unfolded polypeptides which could lead to aggregation. In stark contrast to the threading of unfolded substrates through the Sec translocase, the twin-arginine translocation (Tat) pathway has the unique ability to transport structurally diverse proteins that have already folded in the cytoplasm prior to membrane translocation (examined in ref.8 and elsewhere). The difficulty of this task is usually underscored by the fact that only one other protein transport system in nature, namely the peroxisomal import pathway, is known to exhibit this capability with a similarly diverse set of substrate proteins. The amazing feat of transporting prefolded Tat substrates is performed by a translocase that is completely distinct from your Sec machinery. In alkaline phosphatase (PhoA) altered with a functional Tat transmission peptide was only exported when its native disulfide bonds had been formed to generate the correctly folded molecule.23 In the absence of these bonds, Tat-targeted PhoA was not exported out of the cytoplasm. Hence, not only can the Tat pathway accommodate folded proteins, but it can also discriminate against misfolded proteins. Other proteins whose folding is dependent on the formation of disulfide bonds, such as single-chain Fv (scFv) and FAB antibody fragments, are discriminated in a similar fashion. In fact, the rate of scFv folding is usually a critical determinant of Tat export efficiency, with faster folding scFv antibodies undergoing more efficient translocation than their slower folding counterparts.31 Likewise, thioredoxin-1, a protein that exhibits very fast folding kinetics, is exported by the Tat translocase with very Phellodendrine chloride high efficiency.31 This is in stark contrast to the very inefficient export of thioredoxin-1 when it is fused to a signal peptide that directs post-translational Sec export.32 These observations have led to speculation that Tat export favors folding properties that are diametrically opposite of those required for Sec export. An interesting observation made by two individual groups is usually that Tat-targeted PhoA, which fails to be translocated, is still able to reach the Tat translocase.33,34 This implies that discrimination of the PhoA folding state occurs after targeting to the translocase. In support of this hypothesis, the molecular contacts between misfolded PhoA and the TatBC components of the translocase were notably different from the contacts observed between TatBC and correctly folded PhoA.34 It is possible that these differential contacts reflect active discrimination of folded and mis/unfolded substrates by the Tat translocase. If this interpretation is usually correct, then folding QC would be an inherent property of the Tat translocase. To test this hypothesis, we recently performed a search for genetic Phellodendrine chloride suppressors that inactivate Tat translocase-mediated QC and permit export of the normally export-defective proteins.25 We identified several genetic suppressors that export a misfolded protein called 3B, a designed three-helix-bundle protein that lacks a uniquely folded structure and is thus not tolerated by the wild-type (wt) translocase. Importantly, the isolation of suppressors that inactivated the Tat QC mechanism provides direct evidence for the participation of the Tat translocase.