Supplementary figure 1 Avg. length (AA) c. Conservation of P5 Conservation of P a. Peptides (#) b. WT 75 WT 200 10 **p<0.01 100 Tpn-/- -/- 80 Tpn 150 50 Shared 9 60 100 151 40 Length 8 25 50 20 9 Prevalence (%) Phe or Tyr at P4-P6 (%) P4-P6 at Tyr or Phe Number of peptides of Number 0 7 0 0 -/- -/- -/- WT Tpn WT Tpn WT Tpn MI L V F A T P Amino Acid Length: 8 9 4 n=124 d. 4 n=26 3 3 2 bits 2 WT SAY bits 1 S I VATI NVTI LFPVI EI FVY NLMVI 1RSQVATNGVLFAYI EPRSATFEKDHVYLEQTKRLFMVI 0 1 2 3 4 5 6 7 8 0 2 4 5 6 7 9 1 3 8 N C N C 4 n=7 3 -/- 2 Tpn bits 1 P FPM 0 1 2 3 4 5 6 7 8 N C Supplementary Figure 1. Tapasin-deficient cells present unique H-2Kb peptides, which are longer and lack canonical anchor residues. Kb pMHC I were immunoprecipitated from B6 (WT) and Tpn-/- spleen cells. Eluted peptides were sequenced by mass spectrometry and manually validated. (a) Numbers of peptides specific to WT or Tpn-/- cells or found in both data sets (shared). (b) The average lengths of peptides specific to WT or Tpn-/-!"$" H-2Kb $'$+3$;>'"$?@Db peptides. (d) Logo representation of H-2Kb peptides specific to WT or Tpn-/- cells. Peptides are grouped according to their lengths, and the numbers of included peptides are indicated. The height of each column is proportional to the degree of amino acid conservation and the height of each letter composing the column is proportional to its frequency at the given position. Amino acids are colored as follows: hydrophobic (black), aromatic (purple), acidic (red), basic (blue), neutral (green), and the others (orange). Supplementary Figure 2 a. FSPLNPVRV FVHPGAATVPTM SNLYNHPQLI WAPENAPLKS FAPVAPKFTPV 4000 4000 4000 4000 4000 3000 3000 3000 3000 3000 2000 2000 2000 2000 2000 expression (MFI) expression 1000 1000 1000 1000 1000 b 0 0 0 0 0 H-2D -4 -3 -2 -1 0 1 2 3 4 -4 -3 -2 -1 0 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10-4 10-3 10-2 10-1 100 101 102 103 104 10-4 10-3 10-2 10-1 100 101 102 103 104 10-4 10-3 10-2 10-1 100 101 102 103 104 10 10 10 10 10 10 10 10 10 10 Peptide Concentration (nM) Peptide Concentration (nM) Peptide Concentration (nM) Peptide Concentration (nM) Peptide Concentration (nM) b. FSPLNPVRV FVHPGAATVPTM SNLYNHPQLI WAPENAPLKS FAPVAPKFTPV 100 100 100 100 100 75 75 75 75 75 50 50 50 50 50 expression b 25 25 25 25 25 (relative to 0hr) to (relative H-2D 0 0 0 0 0 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 Recovery Time (hours) Recovery Time (hours) Recovery Time (hours) Recovery Time (hours) Recovery Time (hours) SLNRFIPL ISPYFINT VGQQFGAVGV c. 1000 1000 1000 -/- 750 750 750 Tpn Peptide 500 500 500 LYL8 expression (MFI) expression 250 250 250 b WI9 0 0 0 H-2K 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 Peptide Concentration (nM) Peptide Concentration (nM) Peptide Concentration (nM) d. SLNRFIPL ISPYFINT VGQQFGAVGV 100 100 100 75 75 75 50 50 50 expression 25 25 25 b 3 (relative to 0hr) to (relative 0 0 0 H-2K 0 1 2 3 0 1 2 3 0 1 2 Recovery Time (hours) Recovery Time (hours) Recovery Time (hours) Supplementary Figure 2. Peptides from tapasin-deficient cells form less stable pMHC I on the surface and decay faster. (a). Stabilization of H-2Db MHC I with Tpn-/- peptides. RMA/s cells, incubated overnight to stabilize empty pMHC I on the surface, were incubated with indicated Tpn-/- peptides (top of each graph) or control LTFNYRNL (LYL8, Kb-binding) or WMHHNMDLI (WI9, Db-binding) peptides for 1 hour at 22°C followed by incubation at 37°C for 3 hours prior to analysis by flow cytometry. (b) Decay of H-2Db surface UXZ[\@)Z"[^[X""_` ^"x?@{b. MFI for each sample is normalized to 0hr recovery time. (c) Stability of Kb peptides performed as in (a). (d) Decay of Kb peptides as in (b). Data are representative of three independent experiments (a and c) or contain pooled data from at least three independent experiments (b and d). Data Peptide Accession Set Sequence Length Number Source Protein Name Nuclear factor NF-kappa-B p100 Tpn-/- AALQNLEQL 9 Q9WTK5 subunit C-1-tetrahydrofolate synthase, Tpn-/- AGILNGKLV 9 Q922D8 cytoplasmic Tpn-/- FSHPNVLPV 9 O55222 Integrin-linked protein kinase Heterogeneous nuclear Tpn-/- FSPLNPVRV 9 O35737 ribonucleoprotein H KN motif and ankyrin repeat Tpn-/- GGPENTLVF 9 Q8BX02 domain-containing protein 2 Tpn-/- GINVNAAPF 9 Q60865 Caprin-1 Tpn-/- NSGINVMQV 9 O89090 Transcription factor Sp1 Tpn-/- SSLLSPMSV 9 P80315 T-complex protein 1 subunit delta Tpn-/- YSSENISNF 9 O70400 PDZ and LIM domain protein 1 SAM domain and HD domain- Tpn-/- YVFPGASHN 9 Q60710 containing protein 1 Probable JmjC domain-containing Tpn-/- SLLGGHPRL 9 Q69ZK6 histone demethylation protein 2C Tpn-/- LHLGVTPSVL 10 O89023 Tripeptidyl-peptidase 1 Tpn-/- SNLYNHPQLI 10 Q6NSQ7 Protein LTV1 homolog Tpn-/- VGPLGSTIPM 10 Q9DCP9 DAZ-associated protein 2 Pre-mRNA-processing factor 40 Tpn-/- VMSGMMMSHM 10 Q9R1C7 homolog A V-type proton ATPase subunit B, Tpn-/- VSGVNGPLVI 10 P62814 brain isoform Tpn-/- WAPENAPLKS 10 P18760 Cofilin-1 F-actin-capping protein subunit Tpn-/- AQYNLDQFTPV 11 P47754 alpha-2 Tpn-/- FAPVAPKFTPV 11 Q62523 Zyxin Tpn-/- FAPVNVTTEVK 11 P10126 Elongation factor 1-alpha 1 Trafficking protein particle Tpn-/- LLTSGVEFESL 11 Q3U0M1 complex subunit 9 Glyceraldehyde-3-phosphate Tpn-/- SAPSADAPMFV 11 P16858 dehydrogenase Tpn-/- SLGVGYRTQPM 11 Q8C2Q3 RNA-binding protein 14 Histone-lysine N-methyltransferase Tpn-/- FALQNATGDGKF 12 Q6P2L6 NSD3 Tpn-/- FVHPGAATVPTM 12 Q9DCP9 DAZ-associated protein 2 Tpn-/- SQYGWSGNMERI 12 P08113 Endoplasmin B6 IRMMLEII 8 Q8CHY3 Dymeclin FH1/FH2 domain-containing protein B6 AALLNFDEF 9 Q6P9Q4 1 U3 small nucleolar RNA-interacting B6 AALLNTDLV 9 Q91WM3 protein 2 Tripartite motif-containing B6 AALQNAVAF 9 Q80VI1 protein 56 B6 AALQNLVKI 9 P70168 Importin subunit beta-1 B6 AALVNLDSL 9 Q8CHU3 Epsin-2 Dolichyl-P-Man:Man(7)GlcNAc(2)-PP- dolichyl-alpha-1,6- B6 AAMLNYTHI 9 Q8VDB2 mannosyltransferase tRNA (uracil-5-)-methyltransferase B6 AAPFDTVHI 9 Q8BNV1 homolog A Histone deacetylase complex B6 AAPINTQGL 9 Q8BIH0 subunit SAP130 B6 AAPISPWTM 9 Q3U133 Zinc finger protein 746 Methyltransferase-like protein B6 AAPRSFIFL 9 Q3UIK4 KIAA1627 Dual specificity protein B6 AAPSNLPYL 9 Q8BFV3 phosphatase 4 B6 AAVLNPRFL 9 Q7TPS5 Uncharacterized protein KIAA0528 Heat shock protein 75 kDa, B6 AGTRNIYYL 9 Q9CQN1 mitochondrial B6 AKIQMLDAL 9 Q9D757 Death-associated protein-like 1 Anaphase-promoting complex subunit B6 AMLTNLESL 9 Q8BZQ7 2 B6 AMNVNDLFL 9 P61021 Ras-related protein Rab-5B B6 AMNVNEIFM 9 P35278 Ras-related protein Rab-5C B6 AMYVHAYTL 9 O70435 Proteasome subunit alpha type-3 Histone-lysine N- methyltransferase, H3 lysine-36 B6 ASMINVEYL 9 O88491 and H4 lysine-20 specific Ankyrin repeat domain-containing B6 ASVLNVNHI 9 Q99NH0 protein 17 B6 ASVSNPLFL 9 P70428 Exostosin-2 Signal recognition particle 54 kDa B6 ATIINEEVL 9 P14576 protein B6 ATVSNGPFL 9 Q69ZF8 Male-specific lethal 2 homolog B6 EAVKNLEWI 9 Q02788 Collagen alpha-2(VI) chain B6 ERAQLAAQL 9 P70436 Homeobox protein DLX-4 B6 FAHTNIESL 9 P27773 Protein disulfide-isomerase A3 Phosphatidylinositol-5-phosphate B6 FAIDDHDYL 9 Q91XU3 4-kinase type-2 gamma B6 FAIQNPTLI 9 Q9D2H6 Transcription factor Sp2 Macrophage colony-stimulating B6 FAPKNIYSI 9 P09581 factor 1 receptor B6 FAPYNKPSL 9 Q8R3P2 Protein deltex-2 Eukaryotic translation initiation B6 FGPINSVAF 9 Q9QZD9 factor 3 subunit I Uncharacterized protein CXorf21 B6 FIMTNVDQI 9 Q9D3J9 homolog B6 FSFVNSEFL 9 P68404 Protein kinase C beta type B6 FSLQNQLRL 9 Q9JJ28 Protein flightless-1 homolog ETS-related transcription factor B6 FSPFNPTSL 9 Q9Z2U4 Elf-4 Uncharacterized protein C1orf103 B6 FSVTNPHTM 9 Q8CDD9 homolog B6 FSYLNPQEL 9 Q8C2S5 F-box/LRR-repeat protein 5 Glycerol-3-phosphate dehydrogenase B6 GALKNIVAV 9 Q3ULJ0 1-like protein B6 GAPLNLVDL 9 Q9QWT9 Kinesin-like protein KIFC1 CMP-N-acetylneuraminate-beta- galactosamide-alpha-2,3- B6 GAVKNLTYF 9 Q91Y74 sialyltransferase Interferon-induced very large B6 GMIENGPFL 9 Q80SU7 GTPase 1 B6 GQVINLDQL 9 Q3UVG3 Protein FAM91A1 B6 GSVVNSVAL 9 P59326 YTH domain family protein 1 MAP kinase-activating death domain B6 IAPTNAEVL 9 Q80U28 protein Mitochondrial import inner B6 IAVANAQEL 9 P62075 membrane translocase subunit Tim13 B6 IGPKNYEFL 9 Q8BHY8 Sorting nexin-14 B6 ILHTNLVYL 9 Q62280 Protein SSXT B6 IQIANVTTL 9 Q9CU65 Zinc finger MYM-type protein 2 Peroxisomal carnitine O- B6 IQLMNTAHL 9 Q9DC50 octanoyltransferase B6 IRPGATAVI 9 Q9WTR6 Cystine/glutamate transporter Interferon-inducible double stranded RNA-dependent protein B6 ISPENHISL 9 Q9WTX2 kinase activator A Delta-1-pyrroline-5-carboxylate B6 ISPFNFTAI 9 Q8CHT0 dehydrogenase, mitochondrial B6 ISPVNPVAI 9 Q91WT8 RNA-binding protein 47 B6 KAIVNVIGM 9 Q99NB9 Splicing factor 3B subunit 1 B6 KGVFNVEVV 9 Q8R1A4 Dedicator of cytokinesis protein 7 B6 KSVENFVSL 9 Q8C3J5 Dedicator of cytokinesis protein 2 B6 KSVINTTLV 9 O35114 Lysosome membrane protein 2 B6 LAIRNDEEL 9 Q8CGP5 Histone H2A type 1-F B6 LGLSNLTHL 9 Q9EQU3 Toll-like receptor 9 Conserved oligomeric Golgi complex B6 LLASHRTWI 9 Q921L5 subunit 2 B6 LQLLNTDYL 9 Q9R0L6 Pericentriolar material 1 protein B6 LSHTNILVL 9 Q9EQU3 Toll-like receptor 9 Interferon-inducible double stranded RNA-dependent protein B6 LSLPNTDYI 9 Q9WTX2 kinase activator A B6 LSNINSVFL 9 Q9Z0M6 CD97 antigen B6 MGILNTDTL 9 Q8C4V1 Rho GTPase-activating protein 24 Clathrin coat assembly protein B6 NGVINAAFM 9 Q61548 AP180 B6 NMPWNVDTL 9 Q61081 Hsp90 co-chaperone Cdc37 B6 QPPYNPTYM 9 Q9D7I0 Protein shisa-5 B6 QSLTNILHL 9 Q7TMR0 Lysosomal Pro-X carboxypeptidase Mothers against decapentaplegic