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The signal peptide plus a cluster of positive charges in prion protein dictate chaperone-mediated Sec61 channel gating.

ABSTRACT: The Sec61-complex as a dynamic polypeptide-conducting channel mediates protein transport into the human endoplasmic reticulum (ER) with the help of additional components. ER membrane resident Hsp40-type co-chaperone Sec63 as well as the ER lumenal Hsp70-type chaperone BiP were proposed to facilitate channel opening in a precursor-specific fashion. Here, we report on their rules of engagement in ER import of the prion protein (PrP) by addressing sixteen PrP-related variants which differ in their signal peptides and mature parts, respectively. Transport into the ER of semi-permeabilized human cells was analyzed upon depletion of the components by siRNA- or toxin-treatment. The results are consistent with the view of separate functions of BiP and Sec63 and strongly suggest that the co-chaperone/chaperone-pair facilitates Sec61 channel gating to the open state when precursor polypeptides with weak signal peptides in combination with detrimental features in the adjacent mature part were targeted. Thus, we expand the view of chaperone-mediated Sec61 channel gating by providing a novel example of a polybasic motif that interferes with signal peptide-mediated Sec61 channel gating. This article has an associated First Person interview with the first author of the paper.

SUBMITTER: Ziska A

PROVIDER: S-EPMC6451349 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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The signal peptide plus a cluster of positive charges in prion protein dictate chaperone-mediated Sec61 channel gating.

Ziska Anke A Tatzelt Jörg J Dudek Johanna J Paton Adrienne W AW Paton James C JC Zimmermann Richard R Haßdenteufel Sarah S

Biology open 20190305 3

The Sec61-complex as a dynamic polypeptide-conducting channel mediates protein transport into the human endoplasmic reticulum (ER) with the help of additional components. ER membrane resident Hsp40-type co-chaperone Sec63 as well as the ER lumenal Hsp70-type chaperone BiP were proposed to facilitate channel opening in a precursor-specific fashion. Here, we report on their rules of engagement in ER import of the prion protein (PrP) by addressing sixteen PrP-related variants which differ in their ...[more]

PMID: 30745438

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Project description:Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K+ channel's voltage sensor allosterically modulates opening of its K+-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K+ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K+-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K+-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K+ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors.

Project description:The role of the voltage sensor positive charges in the activation and deactivation gating of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing (by glutamine substitution) and -conserving mutations were constructed in a cDNA encoding the sodium channel alpha subunit that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III-IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. 89:10910-10914.). A total of 16 single and 2 double mutants were constructed and analyzed with respect to voltage dependence and kinetics of activation and deactivation. The most significant effects were observed with substitutions of the fourth positive charge in each domain. Neutralization of the fourth positive charge in domain I or II produced the largest shifts in the voltage dependence of activation, both in the positive direction. This change was accompanied by positive shifts in the voltage dependence of activation and deactivation kinetics. Combining the two mutations resulted in an even larger positive shift in half-maximal activation and a significantly reduced gating valence, together with larger positive shifts in the voltage dependence of activation and deactivation kinetics. In contrast, neutralization of the fourth positive charge in domain III caused a negative shift in the voltage of half-maximal activation, while the charge-conserving mutation resulted in a positive shift. Neutralization of the fourth charge in domain IV did not shift the half-maximal voltage of activation, but the conservative substitution produced a positive shift. These data support the idea that both charge and structure are determinants of function in S4 voltage sensors. Overall, the data supports a working model in which all four S4 segments contribute to voltage-dependent activation of the sodium channel.

Dataset Information

The signal peptide plus a cluster of positive charges in prion protein dictate chaperone-mediated Sec61 channel gating.

Publications

The signal peptide plus a cluster of positive charges in prion protein dictate chaperone-mediated Sec61 channel gating.

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