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. 2017 Jan 19:8:14089.
doi: 10.1038/ncomms14089.

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

Andreas Anderluh  1 , Tina Hofmaier  2 , Enrico Klotzsch  3 , Oliver Kudlacek  2 , Thomas Stockner  2 , Harald H Sitte  2 , Gerhard J Schütz  1
Affiliations

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

Andreas Anderluh et al. Nat Commun. .

Abstract

The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by single-molecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization. After trafficking to the plasma membrane, however, the SERT stoichiometry is fixed. Stabilization of the oligomeric SERT complexes is mediated by the direct binding to phosphoinositide phosphatidylinositol-4,5-biphosphate (PIP2). The observed spatial decoupling of oligomer formation from the site of oligomer operation provides cells with the ability to define protein quaternary structures independent of protein density at the cell surface.

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Figures

Figure 1
Figure 1. Determination of mGFP-SERT oligomer sizes by single molecule brightness analysis.
(a) Thinning out clusters while conserving stoichiometry of labeling (TOCCSL). Using a field stop in the laser beam pathway, a small area of the densely fluorescently labelled cell membrane (i) is irreversibly photobleached (ii, iii). During the recovery phase (iv), SERT oligomers diffuse back into the bleached area. At the onset of this process, they can be discriminated as single, well separated fluorescent spots (v). (b) The oligomeric state of SERT was evaluated in the plasma membrane (left panel) or the endoplasmic reticulum (right panel) by single-molecule brightness analysis. The brightness distributions (in counts) of mGFP–SERT complexes are plotted as pdf. The plots show the distribution of the complexes from the TOCCSL image after the recovery phase (black curves) and the measured brightness of a monomer (red curves); see also Supplementary Fig. 3. A fit yields the distribution of oligomeric sizes at the respective organelle. Scale bars, 10 μm. (c) At the plasma membrane, the mean oligomeric size is independent from the density of SERT (n>22 cells per datapoint; plotted protein densities were 29±17 (s.e.m.) μm−2, 402±31 μm−2 and 840±56 μm−2; s.e.m. of the mean oligomeric sizes were smaller than 0.05). In contrast, at the ER higher expression levels correlate with larger oligomeric sizes (n>19 cells per datapoint; plotted protein densities are 153±13 (s.e.m.) μm−2, 185±24 μm−2, 343±37 μm−2 and 643±36 μm−2; s.e.m. of the mean oligomeric sizes were smaller than 0.05).
Figure 2
Figure 2. Evaluation of the oligomer stability in the plasma membrane and at the ER.
(a) To study the stability of SERT oligomers we performed repeated TOCCSL runs on the same cells (1 run per minute over 10 min), and determined the brightness distributions in each run. Two different scenarios can be distinguished: if oligomers were stable over the time course of the experiment, the total number of diffraction-limited spots would be reduced without altering the brightness distribution (left, scenario i). In contrast, if oligomers would exchange subunits during the 10 min, increasing numbers of mixed SERT oligomers containing both bleached and non-bleached subunits would be observable, thereby shifting the determined oligomeric distribution towards smaller structures (right, scenario ii). (b) Using the repetitive TOCCSL strategy, we have observed no change in the oligomeric distribution of SERT at the plasma membrane (n=20 cells). Oligomeric distributions are shown at the beginning of the experiment (white bars), after 1 min (dark grey), 3 min (light grey), and after 10 min (black). (c) At the ER, however, we observed rearrangement of subunits over the timescale of the experiment, as can be seen by the shift of the distributions towards lower oligomer sizes (n=22 cells). Error bars show the s.e.m.
Figure 3
Figure 3. SERT oligomerization at the plasma membrane depends on PIP2 levels.
(a) We enzymatically depleted PIP2 at the plasma membrane via activation of phospholipase Cγ (PLCγ) by incubating cells for 15 min with the direct PLCγ-activator m-3M3FBS (10 μM). This led to a marked shift of SERT complex sizes towards monomers (dark grey bars). As a negative control, incubation with the inert orthologue o-3M3FBS did not yield any effect (light grey bars) in comparison to the untreated cells (white bars) (n>20 cells per experimental condition). SERT surface densities were similar: 25±14 (s.e.m.) μm−2 (dark grey bars), 38±22 μm−2 (light grey bars), 29±17 μm−2 (white bars). (b) PIP2 depletion via m-3M3FBS resulted in marked dependence of mGFP–SERT oligomerization on mGFP–SERT surface density (n>20 cells per datapoint; plotted protein densities are 25±14 (s.e.m.) μm−2, 48±24 μm−2, 84±17 μm−2, 187±28 μm−2 and 501±40 μm−2; s.e.m. of mean oligomeric sizes were smaller than 0.05). (c) To test for the effect of PIP2 depletion on the stability of the oligomers at the plasma membrane, we performed repetitive TOCCSL runs upon incubating cells with m-3M3FBS (1 μM) (n=19 cells). Now, the SERT complexes showed rapid subunit rearrangement, indicating liberation of SERT oligomers from kinetic trapping. The SERT surface density was 89±21 (s.e.m.) molecules μm−2. Error bars show the s.e.m.
Figure 4
Figure 4. Direct binding of PIP2 to SERT mediates oligomerization.
(a) Analysis of the electrostatic field generated by SERT. The final structure of a 100 ns simulation of a membrane-inserted SERT is shown as viewed from the cytosole (left) or in side-view (right). SERT surface is shown in white, the electrostatic isosurfaces in red (negative potential) and blue (positive potential). For illustration purposes, a PIP2 molecule was placed into the membrane (in space filled representation) in close proximity to the large positively charged area that includes residue K460. (b) We determined the quaternary assembly of the mutant mGFP–SERT–K352A–K460A at the plasma membrane (white bars). Proteins were expressed at a surface density of 83±31 (s.e.m.) molecules μm−2. A distinctive shift to monomers and dimers compared with the wild type (grey bars) was observed (n=23 cells). (c) A pronounced dependence of mean oligomeric state on mGFP–SERT–K352A–K460A surface density was observed, which saturates around 2.8 transporter molecules per oligomer (n>19 cells per datapoint; plotted protein densities are 35±11 (s.e.m.) μm−2, 110±33 μm−2, 237±14 μm−2 and 452±35 μm−2; s.e.m. of mean oligomeric sizes were smaller than 0.05). (d) Repetitive TOCCSL runs revealed rapid protomer exchange kinetics for this mutant (n=18 cells). Error bars show the s.e.m.
Figure 5
Figure 5. A model for PIP2 dependent oligomerization of SERT.
High PIP2 concentrations at the plasma membrane (left) saturates PIP2 binding sites on SERT, impeding further oligomerization of the subunits. Also disassembly of the oligomers is efficiently prevented: in case of PIP2 unbinding, the vacant position is rapidly re-populated by a new PIP2 molecule before the protomers can separate by diffusion. Together, the two effects lead to the kinetic trapping of the oligomeric state at the plasma membrane. At the ER membrane, however, low PIP2 concentrations lead to coexistence of PIP2-ligated and -unligated SERT (right), which are capable of mutual binding. Hence, such conditions enable fast equilibration of the oligomerization process, including subunit exchange between SERT oligomers.
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