1

Supplementary Materials

P. S. Kaeser et al., " RIM1α and RIM1β are synthesized from distinct promoters of the RIM1 gene

to mediate differential but overlapping synaptic functions"

Supplementary Figures

Supplementary Figure 1. RIM1β is a conserved, developmentally regulated isoform of the RIM1

gene. (A) To test whether RIM1β is also expressed in rats, we prepared brain homogenates of two 17

day old Sprague Dawley (SD) rats. We used a polyclonal antiserum against the RIM1 central domain

(top) and a monoclonal antibody against the RIM1 PDZ domain. RIM1α and RIM1αβ KO mice and

littermate controls were used as controls. VCP is used as internal standard. (B) Time course of RIM1α

and RIM1β expression in RIM1α KO mice and wild-type littermate controls at postnatal day 1, 7, 17 and

77. Expression was assessed with a monoclonal anti-RIM1 PDZ domain antibody (top) and a polyclonal

antiserum against the RIM1 central region (2nd from top). Munc18-1 and Rabphilin were used as

presynaptic markers, and GDI and VCP as loading controls.

2

A SPLICE SITE A RIM1α MSSAVGPRGPRPPTV----PPPMQELPDLSHLTEEERNIIMAVMDRQKEEEEKEEAMLKCVVRDMAKPAACKTPRNAESQPHQPPLNIF 85 RIM1β -------------------------- MSHERREKLTEAVPFSSPKAILHTFWSFLSRCVVRDMAKPAACKTPRNAESQPHQPPLNIF 62 RIM2α MSAPLGPRGRPAPTPAASQPPPQPEMPDLSHLTEEERKIIQAVMDRQKKEEEKEQSVLKIKEEHKAQPTQWFPFSGITELVNNVLQPQQ 89 RIM1α β RCVCVPRKPSSEEGGPERDWRLHQQFESYKEQVRKIGEEARRYQGEHKDDAPTCGICHKTKFADGCGHLCSYCRTKFCARCGGRVSLRS 174 RIM1β RCVCVPRKPSSEEGGPERDWRLHQQFESYKEQVRKIGEEARRYQGEHKDDAPTCGICHKTKFADGCGHLCSYCRTKFCARCGGRVSLRS 151 RIM2α KQPNEKEPQT-----------LHQQFEMYKEQVKKMGEES-QQQQEQKGDAPTCGICHKTKFADGCGHNCSYCQTKFCARCGGRVSLRS 166 RIM1α NNEDKVVMWVCNLCRKQQEILTKSGAWFFGSG---PQQPSQDGTLSDTATGAGSE-VPREKKARLQERSRSQTPL---STAAVSSQDT- 255 RIM1β NNEDKVVMWVCNLCRKQQEILTKSGAWFFGSG---PQQPSQDGTLSDTATGAGSE-VPREKKARLQERSRSQTPL---STAAVSSQDT- 232 RIM2α N---KV-MWVCNLCRKQQEILTKSGAWFYNSGSNTPQQPDQK-----ALRGLRSEEAPQEKKAKLHEQTQFQGPPGDSSVPAVERGRAH 246 RIM2β ---------------------------------------------------------------------------MQFETLRQVCNSVL 14 RIM1α ATPGAPLHRNKGAEPSQQAL-GPEQKQASRSRS-EPPRERKKAPGLSEQNGKGGQKSERKRVPKSVVQPGEGIADERERKERRETRRLE 342 RIM1β ATPGAPLHRNKGAEPSQQAL-GPEQKQASRSRS-EPPRERKKAPGLSEQNGKGGQKSERKRVPKSVVQPGEGIADERERKERRETRRLE 319 RIM2α GLTRQDSIKNGSGMKHQIASDMPSDRKRSPSVSRDQNRRYDQSEEREEYSQYVPSDSTMPRSPSDYADRRSQREPQFYEEPDHLNYRDS 335 RIM2β SHFHGVFSSPPNILQNLFGQTLNNARKRSPSVSRDQNRRYDQSEEREEYSQYVPSDSTMPRSPSDYADRRSQREPQFYEEPDHLNYRDS 103 RIM1α KGRSQDYSDRP--EKRDNGRVAEDQKQRKEEEYQTRYRSDPNLARYPVKAPPEEQQMRMHARVSRARHERRHSDVALPHTEAAAAAPAE 429 RIM1β KGRSQDYSDRP--EKRDNGRVAEDQKQRKEEEYQTRYRSDPNLARYPVKAPPEEQQMRMHARVSRARHERRHSDVALPHTEAAAAAPAE 429 RIM2α NRRGHRHSKEYIVDDEDVESRDEYERQRREEEYQARYRSDPNLARYPVKPQPYEEQMRIHAEVSRARHERRHSDVSLANAELEDSRISL 424 RIM2β NRRGHRHSKEYIVDDEDVESRDEYERQRREEEYQARYRSDPNLARYPVKPQPYEEQMRIHAEVSRARHERRHSDVSLANAELEDSRISL 192 RIM1α ---------ATAGKRAPATARVSPPESPRARAAAA-----QPPTEHGPPPPRPAPGPAEPPEPRVPEPLRKQGRLDPGSAVLLRKAKRE 504 RIM1β ---------ATAGKRAPATARVSPPESPRARAAAA-----QPPTEHGPPPPRPAPGPAEPPEPRVPEPLRKQGRLDPGSAVLLRKAKRE 481 RIM2α LRMDRPSRQRSVSERRAAMENQRSYSMERTREAQGQSSYPQRTTNHSPPTPRRSPIPLDRPELRRADSLRKQHHLDPSSAV--RKTKRE 511 RIM2β LRMDRPSRQRSVSERRAAMENQRSYSMERTREAQGQSSYPQRTTNHSPPTPRRSPIPLDRPELRRADSLRKQHHLDPSSAV--RKTKRE 279 RIM1α KAESMLRNDSLSSDQSESVRPSPPKPHRPKRGGKRRQMSVSSSEEEGVSTPEYTSCEDVELESESVSEKGDLDYYWLDPATWHSRETSP 593 RIM1β KAESMLRNDSLSSDQSESVRPSPPKPHRPKRGGKRRQMSVSSSEEEGVSTPEYTSCEDVELESESVSEKGDLDYYWLDPATWHSRETSP 570 RIM2α KMETMLRNDSLSSDQSESVRPPPPRPHKSKKGGKMRQVSLSSSEEELASTPEYTSCDDVEIESESVSEKGDMEYSWLEHASWHSSEASP 600 RIM2β KMETMLRNDSLSSDQSESVRPPPPRPHKSKKGGKMRQVSLSSSEEELASTPEYTSCDDVEIESESVSEKGDMEYSWLEHASWHSSEASP 368 RIM1α ISSHPVTWQPSKEGDRLIGRVILNKRT---TMPKESGALLGLKVVGGKMTDLGRLGAFITKVKKGSLADVVGHLRAGDEVLEWNGKPLP 679 RIM1β ISSHPVTWQPSKEGDRLIGRVILNKRT---TMPKESGALLGLKVVGGKMTDLGRLGAFITKVKKGSLADVVGHLRAGDEVLEWNGKPLP 656 RIM2α MSLHPVTWQPSKDGDRLIGRILLNKRLKDGSVPRDSGAMLGLKVVGGKMTESGRLCAFITKVKKGSLADTVGHLRPGDEVLEWNGRLLQ 689 RIM2β MSLHPVTWQPSKDGDRLIGRILLNKRLKDGSVPRDSGAMLGLKVVGGKMTESGRLCAFITKVKKGSLADTVGHLRPGDEVLEWNGRLLQ 457 RIM1α GATNEEVYNIILESKSEPQVEIIVSRPIGDIPRIPESSHPPLESSSSSFESQKMERPSISVISPTSPGALKDAPQVLPGQLS------- 761 RIM1β GATNEEVYNIILESKSEPQVEIIVSRPIGDIPRIPESSHPPLESSSSSFESQKMERPSISVISPTSPGALKDAPQVLPGQLS------- 738 RIM2α GATFEEVYNIILESKPEPQVELVVSRPIGDMPRIPDSTHAQLESSSSSFESQKMDRPSISVTSPMSPGMLRDVPQFLSGQLSSQSLSRR 778 RIM2β GATFEEVYNIILESKPEPQVELVVSRPIGDMPRIPDSTHAQLESSSSSFESQKMDRPSISVTSPMSPGMLRDVPQFLSGQLSSQSLSRR 546 RIM1α ---------VKLWYDKVGHQLIVNVLQATDLPPRVDGRPRNPYVKMYFLPDRSDKSKRRTKTVKKLLEPKWNQTFVYSHVHRRDFRERM 841 RIM1β ---------VKLWYDKVGHQLIVNVLQATDLPPRVDGRPRNPYVKMYFLPDRSDKSKRRTKTVKKLLEPKWNQTFVYSHVHRRDFRERM 818 RIM2α TTPFVPRVQIKLWFDKVGHQLIVTILGAKDLPSREDGRPRNPYVKIYFLPDRSDKNKRRTKTVKKTLEPKWNQTFIYSPVHRREFRERM 867 RIM2β TTPFVPRVQIKLWFDKVGHQLIVTILGAKDLPSREDGRPRNPYVKIYFLPDRSDKNKRRTKTVKKTLEPKWNQTFIYSPVHRREFRERM 635 RIM1α LEITVWDQPRVQDEESEFLGEILIELETALLDDEPHWYKLQTHDESSLPLPQPSPFMPRRHIHGESSSKKLQRSQRISDSDISDYEVDD 930 RIM1β LEITVWDQPRVQDEESEFLGEILIELETALLDDEPHWYKLQTHDESSLPLPQPSPFMPRRHIHGESSSKKLQRSQRISDSDISDYEVDD 907 RIM2α LEITLWDQARVREEESEFLGEILIELETALLDDEPHWYKLQTHDVSSLPLPHPSPYMPRRQLHGESPTRRLQRSKRISDSEVSDYDCED 956 RIM2β LEITLWDQARVREEESEFLGEILIELETALLDDEPHWYKLQTHDVSSLPLPHPSPYMPRRQLHGESPTRRLQRSKRISDSEVSDYDCED 724

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RIM1α GIGVVPPVGYRASARESKATTLTVPEQQRTTHHRSRSVSPHRGDDQGRPRSRLPNVPL-QRSLDEIHPTRRSRSPTRHHDASRSPADHR 1018 RIM1β GIGVVPPVGYRASARESKATTLTVPEQQRTTHHRSRSVSPHRGDDQGRPRSRLPNVPL-QRSLDEIHPTRRSRSPTRHHDASRSPADHR 995 RIM2α GVGVVS--DYRHDGRDLQSSTLSVPEQVMSSNHCSPSGSPHRVDVIGRTRSWSPSVPPPQRNV-E-QGLRGTRATGHYNTISRMD-RHR 1040 RIM2β GVGVVS--DYRHDGRDLQSSTLSVPEQVMSSNHCSPSGSPHRVDVIGRTRSWSPSVPPPQRNV-E-QGLRGTRATGHYNTISRMD-RHR 808 SPLICE SITE B RIM1α SRHVESQYSSEPDSELLMLPRAKRGRSAESLHMTSELQPSLDRARSASTNCLRPDTSLHSPERERHSRKSERCSIQKQSRKGTASDADR 1107 RIM1β SRHVESQYSSEPDSELLMLPRAKRGRSAESLHMTSELQPSLDRARSASTNCLRPDTSLHSPERERHSRKSERCSIQKQSRKGTASDADR 1084 RIM2α V--MDDHYSSERDSHFLTLPRSRHRQTSEHHHRDG-----------------------------------------------------R 1074 RIM2β V--MDDHYSSERDSHFLTLPRSRHRQTSEHHHRDG-----------------------------------------------------R 842 RIM1α VLPPCLSRRGYATPRATDQPVVRGKYPTRSRSSEHSSVRTLCSMHHLAPGGSAPPSPLLTRTHRQGSPTQSPPADTSFGSRRGRQLPQV 1196 RIM1β VLPPCLSRRGYATPRATDQPVVRGKYPTRSRSSEHSSVRTLCSMHHLAPGGSAPPSPLLTRTHRQGSPTQSPPADTSFGSRRGRQLPQV 1173 RIM2α -DCEAADRQPYHRSRSTEQRPLLERTTTRSRSSERADTNLMRSMPSLMTGRSAPPSPALSRSHPRTGSVQTSPSSTPVTGRRGRQLPQL 1162 RIM2β -DCEAADRQPYHRSRSTEQRPLLERTTTRSRSSERADTNLMRSMPSLMTGRSAPPSPALSRSHPRTGSVQTSPSSTPVTGRRGRQLPQL 930 RIM1α PVRSGSIEQ--------------ASLVVEERTRQMKVKVHRFKQTTGSGSSQELDHEQYSKYNIHKDQYRSCDNASAKSSDSDVSDVSA 1271 RIM1β PVRSGSIEQ--------------ASLVVEERTRQMKVKVHRFKQTTGSGSSQELDHEQYSKYNIHKDQYRSCDNASAKSSDSDVSDVSA 1248 RIM2α PPK-GTLERMITEDMDSTRKRNSGAMDIEERNRQMK--LNKYKQVAGS--DPRLEQDYHSKYRSGWDPHRGADTVSTKSSDSDVSDVSA 1246 RIM2β PPK-GTLERMITEDMDSTRKRNSGAMDIEERNRQMK--LNKYKQVAGS--DPRLEQDYHSKYRSGWDPHRGADTVSTKSSDSDVSDVSA 1014 SPLICE SITE C RIM1α ISRASSTSRLSSTSFMSEQSERPRG-R-ISSFTPKMQGRRMGTSGRAIIKSTSVSGEIYTLERNDGSQSDTAVGTVGAGGKKRRSSLSA 1358 RIM1β ISRASSTSRLSSTSFMSEQSERPRG-R-ISSFTPKMQGRRMGTSGRAIIKSTSVSGEIYTLERNDGSQSDTAVGTVGAGGKKRRSSLSA 1335 RIM2α VSRTSSASRFSSTSYMSVQSERPRGNRKISVFTSKMQSRQMGVSGKSMAKSTSISGDMCSLEKNDGSQSDTAVGALGTSGKKRRSSIGA 1335 RIM2β VSRTSSASRFSSTSYMSVQSERPRGNRKISVFTSKMQSRQMGVSGKSMAKSTSISGDMCSLEKNDGSQSDTAVGALGTSGKKRRSSIGA 1103 RIM2γ ------------------- MGRQGLGGTGAAGRSMQRSQS--------------RSSLSA 27 RIM3γ -------MFNGEPGPASAGASRNVVRSSSISGEICGSQQ-AGGGAGTTTAKKRRSSLGA 51 RIM4γ -----------MERSQS--------------RLSLSA 12 RIM1α KVVAIV--SRRSRSTSQLSQTESGHKKLKSTIQRSTETGMAAEMR-KMVRQPSRESTDGSINSYSSEGNLIFPGVRVGPDSQFSDFLDG 1444 RIM1β KVVAIV--SRRSRSTSQLSQTESGHKKLKSTIQRSTETGMAAEMR-KMVRQPSRESTDGSINSYSSEGNLIFPGVRVGPDSQFSDFLDG 1421 RIM2α KMVAIVGLSRKSRSASQLSQTEGGGKKLRSTVQRSTETGLAVEMRNWMTRQASRESTDGSMNSYSSEGNLIFPGVRLASDSQFSDFLDG 1424 RIM2β KMVAIVGLSRKSRSASQLSQTEGGGKKLRSTVQRSTETGLAVEMRNWMTRQASRESTDGSMNSYSSEGNLIFPGVRLASDSQFSDFLDG 1192 RIM2γ SFEALAGYFPCMNSLEE-DEGEGGGKKLRSTVQRSTETGLAVEMRNWMTRQASRESTDGSMNSYSSEGNLIFPGVRLASDSQFSDFLDG 115 RIM3γ KMVAIVGLTQWSKSTLQLPQPEGATKKLRSNIRRSTETGIAVEMRSRVTRQGSRESTDGSTNSNSSEGTFIFP-TRLGAESQFSDFLDG 139 RIM4γ SFEALAIYFPCMNSFDD--EDAADSRRLKGAIQRSTETGLAVEMPSRTLRQASHESIEDSMNSYGSEGNLNYGGVCLASDAQFSDFLGS 99 RIM1α LGPAQLVGRQTLATPAMGDIQIGMEDKKGQLEVEVIRARSLTQKPGSKSTPAPYVKVYLLENGACIAKKKTRIARKTLDPLYQQSLVFD 1533 RIM1β LGPAQLVGRQTLATPAMGDIQIGMEDKKGQLEVEVIRARSLTQKPGSKSTPAPYVKVYLLENGACIAKKKTRIARKTLDPLYQQSLVFD 1510 RIM2α LGPAQLVGRQTLATPAMGDIQVGMMDKKGQLEVEIIRARGLVVKPGSKTLPAPYVKVYLLDNGVCIAKKKTKVARKTLEPLYQQLLSFE 1513 RIM2β LGPAQLVGRQTLATPAMGDIQVGMMDKKGQLEVEIIRARGLVVKPGSKTLPAPYVKVYLLDNGVCIAKKKTKVARKTLEPLYQQLLSFE 1281 RIM2γ LGPAQLVGRQTLATPAMGDIQVGMMDKKGQLEVEIIRARGLVVKPGSKTLPAPYVKVYLLDNGVCIAKKKTKVARKTLEPLYQQLLSFE 204 RIM3γ LGPAQIVGRQTLATPPMGDVHIAIMDRSGQLEVEVIEARGLTPKPGSKSLPATYIKAYLLENGACLAKKKTKVAKKTCDPLYQQALLFD 228 RIM4γ MGPAQFVGRQTLATTPMGDVEIGLQERNGQLEVDIIQARGLTAKPGSKTLPAAYIKAYLLENGICIAKKKTKVARKSLDPLYNQVLLFP 188 RIM1α ESPQGKVLQVIVWGDYGRMDHKCFMGVAQILLEELDLSSMVIGWYKLFPPSSLVDPTLAPLTRRASQSSLESSSGPPCIRS* 1614 RIM1β ESPQGKVLQVIVWGDYGRMDHKCFMGVAQILLEELDLSSMVIGWYKLFPPSSLVDPTLAPLTRRASQSSLESSSGPPCIRS* 1591 RIM2α ESPQGKVLQIIVWGDYGRMDHKSFMGVAQILLDELELSNMVIGWFKLFPPSSLVDPTLAPLTRRASQSSLESSTGPSYSRS* 1594 RIM2β ESPQGKVLQIIVWGDYGRMDHKSFMGVAQILLDELELSNMVIGWFKLFPPSSLVDPTLAPLTRRASQSSLESSTGPSYSRS* 1362 RIM2γ ESPQGKVLQIIVWGDYGRMDHKSFMGVAQILLDELELSNMVIGWFKLFPPSSLVDPTLAPLTRRASQSSLESSTGPSYSRS* 285 RIM3γ EGPQGKVLQVIVWGDYGRMDHKCFMGMAQIMLDELDLSAVVTGWYKLFPTSSVADSTLGSLTRRLSQSSLESATSP--SCS* 307 RIM4γ ESPQGKVLQVIVWGNYGRMERKQFMGVARVLLEELDLTTLAVGWYKLFPTSSMVDPATGPLLRQASQLSLESTVGPCGERS* 269

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B Exon1 RIM1α N-terminus human MSSAVGPRGPRPPTVPPPMQELPDLSHLTEEERNIIMA-VMDRQKEEEEKEEAMLK⏐RLHQQFESYKEQVRKIGEEARRYQGEHKDDAPTCGIC rat MSSAVGPRGPRPPTVPPPMQELPDLSHLTEEERNIIMA-VMDRQKEEEEKEEAMLK⏐RLHQQFESYKEQVRKIGEEARRYQGEHKDDAPTCGIC mouse MSSAVGPRGPRPPTVPPPMQELPDLSHLTEEERNIIMA-VMDRQKEEEEKEEAMLK⏐RLHQQFESYKEQVRKIGEEARRYQGEHKDDAPTCGIC worm ---------------MDDPSMMPDLSHLSAEEREIIEN⏐VFKRQKDEEAKETQISQ⏐KASEELSELDKQITERKETSKKLVGT-QDDA-ICQIC fly ---------------MD---EMPDLSHLTPHERMQIEN-VLMRQKQEEEKQNEIMR⏐RKQDEVVTLEMQIRQRSEQQKKA-GV-ELDA-TCHIC

C Exon1 RIM1β human ----------MQCSFPSPKAILHGIWSFQNN⏐RLHQQFESYKEQVRKIGEEARRYQGEHKDDAPTCGIC mouse MSHERREKLTEAVPFSSPKAILHTFWSFLSR⏐RLHQQFESYKEQVRKIGEEARRYQGEHKDDAPTCGIC

Supplementary Figure 2. Protein alignment of RIM1β with the known RIM isoforms. (A) Full

length alignment of all known RIM isoforms in rat. RIM1β exists in the same splice variants as RIM1α

at splice site A. The alignment is based on rat sequences, but the protein sequence of the RIM1β specific

exon is derived from mouse. We could not identify the rat exon because the rat genomic database is still

incomplete. (B) Alignment of the RIM1α N-termini corresponding to exon 1’ and parts of exon 4 from

various species. For simplicity, alternatively spliced exons 2 and 3 in human, rat and mouse are omitted.

⏐marks exon borders. (C) Alignment of the RIM1β N-termini corresponding to exon 1’’ and parts of

exon 4 from human and mouse. We found a human genomic sequence (gi14018252, RP11-161N21) on

chromosome 6 that contains an exon that was conserved to 57% compared to mouse RIM1β and is

located 127.5 kb upstream of exon1’ of human RIM1α on chromosome 6. Corresponding exons in D.

melanogaster and C. elegans could not be found by database analysis. For simplicity, alternatively

spliced exons 2 and 3 are omitted. ⏐marks exon borders.

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Supplementary Figure 3. Generation of the conditional RIM1αβ KO mice. (A) Wild-type and

mutant allele of the original RIM1αβ knock-in line after homologous recombination. (NR, neomycin

resistance cassette; OP, outside probe; N, NcoI was used as 5’ Southern site; K, KpnI was used as 3’

6

Southern site). Exon 6 encodes for the linker sequence between the RIM1 zinc finger and the RIM1 PDZ

domain that contains the serine 413 protein kinase A phosphorylation site. The targeting construct

contained a serine 413 to alanine point mutation to test the in vivo function of this phosphorylation site

(Kaeser et al., 2008). (B) Protein and DNA sequence showing wild type RIM1 and the original sequence

of the targeting vector where the serine 413 to alanine point mutation was inserted. SphI and BglI sites

were introduced into the exon with the point mutation. (C) Southern blotting of the embryonic stem cell

clones with the 5’ outside probe after NcoI digestion. The wild-type band has a size of 8.0 kilobases

(kb), the mutant band 3.8 kb. 5B12 is the clone that was used for generating the mice. (D) Southern

blotting of expanded embryonic stem cell clones with the 3’ outside probe after KpnI digest. The wild-

type band has an expected size if 12.5 kb, the mutant band is 16.5 kb. (E) PCR amplification of the

expanded embryonic stem cell clones over the loxP recombination site 5’ of exon 6 (top), and over the

serine 413 to alanine point mutation with consecutive BglI digest (bottom, M, marker). In clone 5B12,

the BglI site that was introduced with the point mutation has disappeared, but homologous

recombination was shown by Southern blotting on the 5’ and the 3’ side. (F) Sequencing result after

PCR amplification of exon 6 from 2 embryonic stem cell clones. Clone 3G12 contains the mutation, in

clone 5B12 the DNA sequence is repaired back to wild-type (the sense primer annealed to the 5’ loxp

site and the antisense primer to the 3’ end of exon 6). Together C to F demonstrate that clone 5B12 is

homologously recombined, but the serine 413 point mutation, the BglI site and SphI site that were

introduced into exon 6 have been corrected by a mismatch repair mechanism in embryonic stem cells

(Steeg et al., 1990; Maximov et al., 2008).

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Supplementary Figure 4. No N-terminal zinc finger fragment is detectable in the RIM1αβ KO

mice. Western blotting with an antibody that recognizes the RIM1α N-terminus fails to detect a short N-

terminal fragment in the RIM1αβ KO mice. RIM1α KO mice and wild-type littermate mice were used as

controls. A 7.5% gel is shown on top, a 13% gel shown on the bottom. The top panel is derived from the

same experiment as shown in Figure 2D. An antibody against VCP was used as a loading control. *;

cross-reactive band with rabphilin (Schoch et al., 2002), **; faint cross-reactive band of unknown origin.

8

9

Supplementary Figure 5. Complete protein quantitations in S2 and P2 fractions of RIM1αβ KO

mice. (A) Western blotting of P2 and S2 fractions with 125iodine labeled secondary antibodies for

multiple active zone and other proteins in brains from RIM1αβ KO mice and wild-type littermate

controls (n = 3 for each genotype, all at 8-9 weeks of age, * marks SNAP-25, this antibody was added at

the same time, but not used for quantitation). β-actin, GDI and VCP are shown as a loading controls and

were used for normalization in P2 and S2 fractions. (B, C) Quantitative analysis of protein contents in

P2 (B) or S2 (C) normalized to VCP, GDI or β-actin. (D) Percent solubility expressed as S2/(P2+S2),

solubility is not indicated when less than 3% of the total amount of protein were soluble (statistical

significance in B-D: * p < 0.05, ** p < 0.01, ***, p < 0.005). In addition to the changes that are

discussed in the main text, there was a small but significant increase in the solubility of complexin I. It is

unclear how this change in solubility of complexin I relates to the deletion of RIM1αβ.

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Supplementary Figure 6. Kinetic analysis of miniature release at inhibitory syanpses in area CA1

of the hippocmapus. (A) Representative, averaged traces (top) and kinetic analysis (bottom) of mIPSCs

from RIM1α KO mice and wild-type littermates. Rise time (10-90%) and decay time constant (τ)

summary plots are derived from at least 300 mIPSCs for each panel. (B) Representative, averaged traces

and kinetic analysis from RIM1αβ KO and littermate control mice. The values and statistical data of this

experiment can be found in supplementary table 4.

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Supplementary tables

Supplementary table 1. Numerical values of quantitative analysis of RIM1β expression in wild-type

and RIM1α KO mice. These numbers correspond to Fig. 1C and D of the manuscript.

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Mouse line

+/+ +/- or +/f -/- or f/f number p-value

RIM1αβfloxed 26.3% 49.1% 24.6% 114 0.95 before background mixing

RIM1α KO* 25.0% 55.0% 20.0% 695 0.01 RIM1αβ KO 29.5% 64.0% 6.5% 430 1 x 10-17

after background mixing RIM1α KO 29.3% 48.8% 21.9% 123 0.49

RIM1αβ KO 34.1% 56.4% 9.5% 126 0.0005

Supplementary table 2. Survival of mouse lines before and after background mixing for 2 generations.

*, these numbers are taken from (Schoch et al., 2002). The numbers indicated correspond to Fig. 3A and

B in the manuscript. Alleles: +, wild-type; f, floxed knock-in; -, KO.

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A

B

14

C

Supplementary table 3. Protein quantitation in P2 and S2. Numerical values for the proteins that

were quantified are given for the insoluble P2 fraction (A), the soluble S2 fraction (B) and the percent

solubility (C). These values correspond to Fig. 4 and Supplementary Fig. 5. Significant results are

shown in bold.

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Parameter Mouse line gt Value n* p-value Statistical test Fig. PPF, 10 ms RIM1αb KO +/+

-/- 1.14 ± 0.08 1.61 ± 0.05

4/5 4/6

< 0.0001 Student’s t-test 5A

PPF, 40 ms RIM1αb KO +/+ -/-

1.49 ± 0.08 1.82 ± 0.05

4/5 4/6

< 0.005 Student’s t-test 5A

PPF, 80 ms RIM1αb KO +/+ -/-

1.46 ± 0.07 1.72 ± 0.03

4/5 4/6

< 0.005 Student’s t-test 5A

PPF, 200 ms RIM1αb KO +/+ -/-

1.29 ± 0.04 1.49 ± 0.02

4/5 4/6

< 0.001 Student’s t-test 5A

Train, 5th amplitude

RIM1αb KO +/+ -/-

117.2 ± 6.72% 202.0 ± 12.8%

3/4 3/5

< 0.0001 Student’s t-test 5B

Train, 15th amplitude

RIM1αb KO +/+ -/-

102.7 ± 3.03% 219.1 ± 18.0%

3/4 3/5

< 0.0001 Student’s t-test 5B

Train, 25th amplitude

RIM1αb KO +/+ -/-

83.6 ± 4.76% 210.4 ± 21.6%

3/4 3/5

< 0.0001 Student’s t-test 5B

MK-801, τ (stimulus #)

RIM1αb KO +/+ -/-

16.4 ± 1.9 38.0 ± 3.8

3/9 3/9

< 0.0001 Student’s t-test 5C

mIPSC frequency

RIM1α KO +/+ -/-

6.60 ± 0.87 Hz 3.76 ± 0.68 Hz

2/6 2/7

< 0.05 Student’s t-test 6A

mIPSC amplitude

RIM1α KO +/+ -/-

11.50 ± 0.68 pA 11.50 ± 0.42 pA

2/6 2/7

> 0.5 Student’s t-test 6A

mIPSC frequency

RIM1αβ KO +/+ -/-

6.52 ± 0.87 Hz 2.72 ± 0.40 Hz

2/7 2/9

< 0.001 Student’s t-test 6B

mIPSC amplitude

RIM1αβ KO +/+ -/-

11.70 ± 0.59 pA 12.90 ± 0.43 pA

2/7 2/9

> 0.1 Student’s t-test 6B

mIPSC rise time

RIM1α KO +/+ -/-

1.37 ± 0.19 ms 1.30 ± 0.11 ms

2/6 2/7

> 0.5 Student’s t-test Sup.6A

mIPSC τ RIM1α KO +/+ -/-

17.8 ± 1.3 ms 18.7 ± 2.0 ms

2/6 2/7

> 0.5 Student’s t-test Sup.6A

mIPSC rise time

RIM1αβ KO +/+ -/-

1.37 ± 0.12 ms 1.35 ± 0.15 ms

2/7 2/9

> 0.5 Student’s t-test Sup.6B

mIPSC τ RIM1αβ KO +/+ -/-

17.9 ± 1.8 ms 18.6 ± 2.4 ms

2/7 2/9

> 0.5 Student’s t-test Sup.6B

i/o curve, 6V

RIM1α KO +/+ -/-

0.53 ± 0.08 nA 0.21 ± 0.05 nA

2/6 2/5

< 0.01

Student’s t-test 6C

i/o curve, 25V

RIM1α KO +/+ -/-

1.46 ± 0.10 nA 0.69 ± 0.12 nA

2/6 2/5

< 0.0001

Student’s t-test 6C

i/o curve, 70V

RIM1α KO +/+ -/-

2.30 ± 0.21 nA 1.12 ± 0.14 nA

2/6 2/5

< 0.00001

Student’s t-test 6C

i/o curve, 6V

RIM1αβ KO +/+ -/-

0.59 ± 0.11 nA 0.16 ± 0.02 nA

2/4 2/6

< 0.005

Student’s t-test 6D

i/o curve, 25V

RIM1αβ KO +/+ -/-

1.59 ± 0.14 nA 0.46 ± 0.04 nA

2/4 2/6

< 0.00001

Student’s t-test 6D

i/o curve, 70V

RIM1αβ KO +/+ -/-

2.46 ± 0.28 nA 0.74 ± 0.05 nA

2/4 2/6

< 0.00001

Student’s t-test 6D

PPD, 20 ms RIM1α KO +/+ -/-

0.39 ± 0.03 0.39 ± 0.02

2/6 2/8

> 0.5 Student’s t-test 7A

PPD, 50 ms RIM1α KO +/+ -/-

0.51 ± 0.04 0.52 ± 0.02

2/6 2/8

> 0.5 Student’s t-test 7A

PPD, 200 ms RIM1α KO +/+ -/-

0.69 ± 0.02 0.70 ± 0.01

2/6 2/8

> 0.5 Student’s t-test 7A

16

PPD, 400 ms RIM1α KO +/+ -/-

0.73 ± 0.03 0.74 ± 0.01

2/6 2/8

> 0.5 Student’s t-test 7A

PPD, 20 ms RIM1αβ KO +/+ -/-

0.41 ± 0.02 0.54 ± 0.03

4/9 5/10

< 0.005 Student’s t-test 7B

PPD,50 ms RIM1αβ KO +/+ -/-

0.52 ± 0.03 0.62 ± 0.02

4/9 5/10

< 0.005 Student’s t-test 7B

PPD, 200 ms RIM1αβ KO +/+ -/-

0.67 ± 0.02 0.76 ± 0.01

4/9 5/10

< 0.001 Student’s t-test 7B

PPD, 400 ms RIM1αβ KO +/+ -/-

0.72 ± 0.02 0.78 ± 0.01

4/9 5/10

< 0.05 Student’s t-test 7B

Mossy fiber LTP

RIM1αb KO +/+ -/-

173 ± 2.9% 101 ± 1.3%

2/3 2/3

< 0.00001 > 0.05

One-way ANOVA, before vs. after induction

9A

I-LTD RIM1αb KO +/+ -/-

82.3 ± 3.8% 97.6 ± 3.8%

4/7 4/7

< 0.001 > 0.5

One-way ANOVA, before vs. after induction

9B

Supplementary table 4. Numerical values for electrophysiological analyses in acute brain slices. gt;

genotype, n*; number of animals/slices.

17

Parameter Mouse line gt Value n* p-value Statistical test Fig. IPSC amplitude

RIM1α KO +/- -/-

3.78 ± 0.56 nA 2.18 ± 0.31 nA

2/9 2/11

< 0.05 Student’s t-test 8A

IPSC charge RIM1α KO +/- -/-

0.30 ± 0.06 nC 0.13 ± 0.02 nC

2/9 2/11

< 0.01 Student’s t-test 8A

IPSC amplitude

f RIM1αβ Δcre cre

3.91 ± 0.28 nA 2.10 ± 0.19 nA

4/22 4/24

< 0.001 Student’s t-test 8B

IPSC charge f RIM1αβ Δcre cre

0.46 ± 0.04 nC 0.18 ± 0.02 nC

4/22 4/24

< 0.001 Student’s t-test 8B

PPD, 25 ms RIM1α KO +/- -/-

0.56 ± 0.08 0.85 ± 0.11

2/9 2/9

= 0.052 Student’s t-test 8C

PPD, 50 ms RIM1α KO +/- -/-

0.69 ± 0.08 0.96 ± 0.08

2/9 2/9

= 0.032 Student’s t-test 8C

PPD,100 ms RIM1α KO +/- -/-

0.84 ± 0.06 1.06 ± 0.07

2/9 2/9

= 0.033 Student’s t-test 8C

PPD,200 ms RIM1α KO +/- -/-

0.94 ± 0.04 0.95 ± 0.07

2/9 2/9

= 0.909 Student’s t-test 8C

PPD,500 ms RIM1α KO +/- -/-

0.91 ± 0.02 0.96 ± 0.06

2/9 2/9

= 0.443 Student’s t-test 8C

PPD, 1 s RIM1α KO +/- -/-

0.94 ± 0.02 0.92 ± 0.05

2/9 2/9

= 0.687 Student’s t-test 8C

PPD, 25 ms f RIM1αβ Δcre cre

0.33 ± 0.03 0.67 ± 0.06

3/12 3/15

= 0.00015 Student’s t-test 8D

PPD, 50 ms f RIM1αβ Δcre cre

0.43 ± 0.03 0.74 ± 0.05

3/12 3/15

= 0.000033 Student’s t-test 8D

PPD,100 ms f RIM1αβ Δcre cre

0.57 ± 0.02 0.81 ± 0.03

3/12 3/15

= 0.000012 Student’s t-test 8D

PPD,200 ms f RIM1αβ Δcre cre

0.76 ± 0.03 0.89 ± 0.03

3/12 3/15

= 0.011 Student’s t-test 8D

PPD,500 ms f RIM1αβ Δcre cre

0.84 ± 0.02 0.88 ± 0.02

3/12 3/15

= 0.165 Student’s t-test 8D

PPD, 1 s f RIM1αβ Δcre cre

0.85 ± 0.02 0.90 ± 0.02

3/12 3/15

= 0.063 Student’s t-test 8D

Supplementary table 5. Numerical values for electrophysiological analyses in cultured hippocampal

neurons. gt; genotype, n*; number of cultures/neurons.

18

Supplementary References

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Sudhof TC (2008) RIM1alpha phosphorylation at serine-413 by protein kinase A is not required for

presynaptic long-term plasticity or learning. Proc Natl Acad Sci U S A 105:14680-14685.

Maximov A, Lao Y, Li H, Chen X, Rizo J, Sorensen JB, Sudhof TC (2008) Genetic analysis of

synaptotagmin-7 function in synaptic vesicle exocytosis. Proc Natl Acad Sci U S A 105:3986-3991.

Schoch S, Castillo PE, Jo T, Mukherjee K, Geppert M, Wang Y, Schmitz F, Malenka RC, Sudhof TC (2002)

RIM1alpha forms a protein scaffold for regulating neurotransmitter release at the active zone. Nature

415:321-326.

Steeg CM, Ellis J, Bernstein A (1990) Introduction of specific point mutations into RNA polymerase II by

gene targeting in mouse embryonic stem cells: evidence for a DNA mismatch repair mechanism. Proc

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