SERT. Synphys. VAMP2/Synbrev. Syngyr VMAT2 SCAMP1. SNAP25 NMDA Glt. M6a

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1 Supplementary Figure S1 kda H P1 P2 LP1 LP SERT Synphys VAMP2/Synbrev Syngyr VMAT2 SCAMP1 SNAP25 NMDA Glt M6a Figure S1: Subcellular fractionation of mouse brain tissue was carried out similar to rat brain tissue as described in the Method section. Resident synaptic vesicle proteins, such as synaptophysin, synaptobrevin (VAMP2) or the vesicular monoamine transporter VMAT2, show enrichment in the LP2 fraction relative to P2 and are less abundant in LP1 (compare to Fig. 4A). Typical plasma membrane proteins, such as the NMDA receptor and Glt, are de-enriched in LP2 relative to P2, and more abundant in LP1. SERT and the SERT interacting protein GPM6a are both detected at similar level in P2 and LP2.

2 Supplementary Figure S2 Supplementary Figure 2. Ultrastructural localization of SERT in rat hippocampus. A: Electron micrograph illustrates axonal (a, longitudinal section) localization of SERT (10 nm immunogold particles, forked arrowhead) in the CA1 area of the rat hippocampus. Note that the gold labeling is in close apposition to the mitochondrial membrane (m). Scale bar, A: 500 nm. B: Serotonergic axon (a) showing immunogold labeling (10 nm gold; forked arrowheads) for SERT found in cytoplasmic structures and occasionally on axonal membrane in CA1 area of rat hippocampus. Axon terminal (at) making symmetric contact to the dendritic shaft (d) was not labelled. C: A serotonergic terminal (at) forming symmetric contact onto the dendritic shaft (d) in the CA1 area of rat hippocampus. The postembedding immunogold labeling for SERT (10 nm gold; forked arrowheads) was found at lower level presynaptically on synaptic vesicle membrane and in the plasma membrane of the axon terminal. Forked arrowhead shows also gold particle on presynaptic membrane in axon terminal (at) making asymmetric contact to the spine (s). D: Serotonergic axon terminal (at) making symmetric contact with the dendritic shaft (d) in CA1 area of the rat hippocampus. Forked arrowheads indicate immunogold labeling for SERT (10 nm gold) found on the membrane of synaptic vesicles. Glutamatergic axon terminal (at) making asymmetric contact to the spine (s) was not labeled. Scale bar, B-D: 200 nm. Abbreviations: a, axon; at, axon terminal; d, dendritic shaft; m, mitochondrion; s, spine.

3 Supplementary Figure S3 kda Rat Mouse WT WT ko Supplementary Figure S3: Western blot analysis verification of SERT antibody specificity. P2 membrane fractions were prepared from frozen wildtype and SERT knockout mouse brain, as well as wildtype rat brain, and loaded onto a 10% SDS polyacrylamide gel as indicated. Western blot analysis was carried out using rabbit anti-sert antibody (Synaptic Systems) and fluorescence labelled secondary IgG antibody DyLight 800 Goat anti-rabbit (gree, Thermo Scientific). The membrane was scanned using the LICOR Odyssey system (LICOR Biosciences). [This figure was generated by Eric Brown, University College Dublin; SERT knockout brain tissue was a generous gift of Harald Sitte, University of Vienna]

4 Supplementary Table S1: Extended mass spectrometry data Affinity purification run 1 Affinity purification run 2 GST-CSERT GST-N100-SERT GST-N115-SERT Protein_ID Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov AT1B T PPIA GPM6B CNTN AT1B FYN CNRP PGM STXB TBB2A ACTB EAA GBB TBB TBA4A GRP VPP SYT DPYL TBB AT2B GBB AT2B DDAH VAMP Z G3P S6A STX1B GNAI SNP NIPS GPM6A SEPT SHPS ODPA ADT NIPS SV2A CMC ATPO GNAQ STX1A

5 Affinity purification run 1 Affinity purification run 2 GST-CSERT GST-N100-SERT GST-N115-SERT Protein_ID Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov GNAI AT2B COX HCD CH RTN NOMO COF ATP5H TOM CAMKV SNAB VISL S12A ODPB MPCP RAB2A PYGM BDH RAB1B RL RTN SEP B PHB KPYM SYNPO CAZA HPCA PRDX VAPA MPP H2A2B SNAA SDHB PP14C SYN NDUB SNG ODO RAP1B TPIS SIR EVX SYNJ RP3A HSP7C

6 Affinity purification run 1 Affinity purification run 2 GST-CSERT GST-N100-SERT GST-N115-SERT Protein_ID Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov Score #Pep #Uniq %Cov NDUA VDAC DYHC MAP1A COX MCCB PDIA NSF MARCS SCAM SCAM SC24A NOS PP2BA Column definitions Protein_ID Uniprot/Swissprot protein identifier Score protein identification confidence score #Pep number of total peptides contributing to protein identification #Uniq number of unique peptides %Cov percentage of the protein sequence covered by identified peptides