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BackgroundThe serotonin transporter (SERT) is exported from the ERby recruiting SEC24C to its C-terminus [1]. The sameregion also provides a docking site for proteinaceous cha-perones (HSP isoforms) that assist in folding [2]. Based onthese observations, we postulate sequential exchange ofthe chaperone(s) for the COPII coat as a mechanism toprevent premature ER export of partially folded SERT.MethodsSERT (including a series of double and truncation mutantsalong its C-terminus) and related transporters werescreened for their SEC24 isoform dependence, examinedby siRNA-induced SEC24 knock-down in HEK293 cells.The cells were transfected with Sec24 siRNAs and, 48 hlater, with transporter plasmid s (Lipofectamine, Invitro-gen), and the effects were determined by substrate uptakeand confocal microscopy. GST-fusion proteins comprisingthe C-terminus of wild-type and mutant SERTs were usedfor pull-down experiments carried out with SEC24C andSEC24D. To examine the role of heat shock protein (HSP)90 in regulating the formation of COPII vesicles, we trea-ted HEK293 cells expressing wild-type SERT and its RI-607,608-AA mutant (putative ER export motif site) withgeldanamycin, prior to measuring the effect of HSP90inhibition on transporter trafficking.ResultsA lysine residue (K610) residing near the putative ERexport motif on SERT (RI-607,608) was replaced by tyro-sine (Y), the equivalent residue found in NET and DAT,leading to a relaxed preference for SEC24 isoform recruit-ment; SERT-K610Y no longer relied solely on SEC24C,but rather SEC24D for ER export. We searched for HSPisoforms that bound within or close to the SEC24-bindingsite. These experiments revealed a role of HSP90 in relay-ing SERT to SEC24C.ConclusionsWe show that the preference for SEC24 isoforms can bealtered upon mutation of a single residue on the cargoprotein. In SERT, lysine 610 appears to have a role in theinteraction with SEC24C. We base our conclusion on thefollowing evidence: (i) GST pull-downs showing an inter-action with SEC24D, rather than SEC24C, (ii) siRNA-induced knock down of SEC24 isoforms A Dand(iii)co-expression with dominant negative SEC24C/SEC24Dmutants, leading to reduced surface expression of thetransporter.

How does the carboxyl terminus assist folding and ER export of the serotonin transporter?