A Cytosolic Relay of HSP70-1A and HSP90{beta} Monitors the Folding Traȷectory of the Serotonin Transporter [Neurobiology]

September 8th, 2014 by El-Kasaby, A., Koban, F., Sitte, H. H., Freissmuth, M., Sucic, S.

Mutations in the C-terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum (ER). Here we examined the hypothesis that a cytosolic heat-shock protein relay was recruited to the C-terminus to assist folding of SERT. This conjecture was verified by the following observations: (i) the proximal portion of the SERT C-terminus conforms to a canonical binding site for DnaK/HSP70 (heat-shock protein of 70 kDa). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST-fusion protein comprising the C-terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Foerster resonance energy transfer: it was restricted to ER-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed com-plex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP-relay, co-chaperones (e.g., HOP/HSC70-HSP90 organizing protein) were co-immunoprecipitated with the stalled mutants SERT-607RI608-AA and SERT-601PG602-AA. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-607RI608-AA and SERT-601PG602-AA were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.