Supplementary Information

Retrotranslocation
Protein / GI# / Peptides / Remarks / Orientation
Calnexin / 683793 / APVPTGEVYFADSFDR, IADPDAVKPDDWDEDAPSK, KIPNPDFFEDLEPF / Retains incorrectly folded glycoprotein’s in the E.R. / TM
Bip
(GRP78, HSP70-5) / 2506545 / DAGTIAGLNVMR,
DNHLLGTFDLTGIPPAPR, ELEEIVQPIISK,
IEIESFFEGEDFSETLTR,
IINEPTAAAIAYGLDK, ITITNDQNR,
ITPSYVAFTPEGER, LTPEEIER,
NQLTSNPENTVFDAK, SDIDEIVLVGGSTR,
SQIFSTASDNQPTVTIK, TFAPEEISAMVLTK,
TKPYIQVDIGGGQTK, VEIIANDQGNR,
VTHAVVTVPAYFNDAQR, VYEGERPLTK. / E.R. Chaperone / L
PDI
(P55, ERP59) / 129729 / TGPAATTLSDTAAAESLVDSSEVTVIGFFK, VDATEESDLAQQYGVR / Protein dissulfide isomerase / L
Sec61p / None / Detected by WB / TM
Valosin containing protein
(VCP, p97, TER-ATPase, CDC48 ) / 20342005 / EVDIGIPDATGR, LDQLIYIPLPDEK / ND
Ubiquitination
Ubiquitin / 136670 / IQDKEGIPPDQQR, EGIPPDQQR, ESTLHLVLR, TLSDYNIQK, TITLEVEPSDTIENVK / Involved in the ATP-dependant selective degradation of cellular proteins, the maintenance of chromatin structure, the regulation of gene expression, the stress response and ribosome biogenesis. / C
Ubiquitin-activating enzyme E1 / 111228 / None / Detected by WB / C
Ubiquitin-conjugating enzyme E2 N
(Ubiquitin-protein ligase N) / 2501432 / LLAEPVPGIK, TNEAQAIETAR / Catalyse the covalent attachment of ubiquitin to other proteins. / C
Ubiquitin-conjugating enzyme E2, 25 kDa
(Ubiquitin-protein ligase; huntingtin interacting protein 2, HIP-2) / 2507504 / VDLVDENFTELR / Catalyses the covalent attachment of ubiquitin to other proteins. Mediates the selective degradation of short-lived and abnormal proteins. Ubiquitinates huntingtin. / C
Proteasomal degradation
Proteasome subunit alpha type 4 / 4506185 / LLDEVFFSEK / Alpha subunits were detected by WB. Part of a multicatalytic proteinase complex. Involved in ATP/ubiquitin-dependant non-lysosomal proteolytic pathway. / C
Proteasome subunit alpha type 6 / 9910829 / YEAANWK, LYQVEYAFK, AINQGGLTSVAVR, LLDSSTVTHLFK
Proteasome subunit beta type 1 / 16758370 / LSEGFSIHTR
Proteasome subunit beta type 3 / 6755202 / FGPYYTEPVIAGLDPK
Proteasome subunit beta type 5 / 3914434 / FLHGVIVAADSR
LMP-2 / 392959 / None / Detected by WB in INF-g treated cells. LMP2 is one ot the two proteasome subunits encoded by genes in the major histocompatibility comples class II region.
Translocation
TAP
(p115) / 1171952 / DTVIENLR / Detected by WB / ND
Peptide Loading
ERp57 / 16508150 / DGEEAGAYDGPR, DLLTAYYDVDYEK, EATNPPIIQEEKPK, ELNDFISYLQR,FVMQEEFSR,GFPTIYFSPANK / L
Calreticulin
(CRP55, calregulin, HACBP) / 6680836 / DKGLQTSQDAR
EQFLDGDAWTNR
IKDPDAAKPEDWDER
KPEDWDEEMDGEWEPPVIQNPEYK / E.R. chaperone, also found in T cell lytic granules. / L
H-2 Class 1 histocompatibility antigen / 293736 / RPEGDVTLR, WTAADMAAQITR / H2-Kd haplotype / ND

Table 1: Cross presentation processing proteins identified from MS-MS analysis of phagosome. Phagosomes formed by the internalization of latex beads for 60 min, followed by a 60 min chase were isolated. Phagosomes were lysed and proteins were separated by 2D or blue native electrophoresis. Gels obtained were cut, digested with trypsin and the resulting peptides analyzed by nanoLC-MS/MS using a QTOF Ultima™. Peptide, indicates the sequences identified for this protein. Orientation data were based on treatment of isolated phagosomes with pronase. L, lumen; TM, trans-membrane; C, cytoplasm; ND, not determined.

Accession#MW (kDa)PTMDCoverageDescription

(%)

492990132.08059.0Chain A Glutathione S-Transferase

34701970.93016.1Hsp72

1161248972.4608.4Glucose regulated protein 78

83945091.0907.9ubiquitin

2014959483.2807.3Hsp90

1317770035.9806.1Unknown protein for IMAGE:2989740

668030583.3605.8Hsp84

68723993.9205.2Tumor necrosis factor type 1 receptor

associated protein

2007238086.8705.0Similar to HLA-B-associated transcript 3

2852817521.8514.6Similar to inhibin binding protein long

isoform

1671654536.1873.5Vomeronasal 1 receptor C1

892266996.4703.4Hypothetical protein FLJ10786

2084695727.303.3RIKEN cDNA 1110007C24

675500296.0102.8Programmed cell death 6 interacting protein

1284007242.6302.7ISS-homolog to melanoma-associated

antigen

2634299746.3102.2Unnamed protein product

950666955.1502.1Hypothetical protein FLJ20261

47954897.1401.6Glycogen phosphorylase (EC 2.4.1.1)

2505242370.2501.4Similar to SPA-1 like protein p1294

2362380773.4501.4Similar to zinc finger protein 118

1992359598.6701.3Apoptosis related protein APR-5

Restricted database search with non specific cleavage

Accession#MW (kDa)PTMDCoverageDescriptionSequences

(%)

12929342.7503.7OvalbuminpQTAMVLVNAIV

pNVMEERK

Table 2: Protein identification from iterative LC-MS-MS analysis of phagosome-associated polypeptides isolated by affinity for the S5A subunit of the proteasome. Phagosomes formed by the internalization of ovalbumin-opsonized latex beads for 60 min, followed by a 90 min chase in presence of MG-132 to inhibit proteasome activity, were isolated. The polyubiquitinated proteins present in this preparation were isolated by affinity for the S5A subunit of the proteasome. These proteins were then separated by SDS-PAGE on a 4-15% polyacrylamide gradient gel. The upper region of the unstained gel between 75 and 250 kDa, where polyubiquitinated proteins are observed in high amounts (see Fig. 3b), was then cut into 12 slices of about 1 mm. In gel digestion with trypsin was then performed and the resulting peptides analyzed by nanoLC-MS/MS using a QTOF Ultima™. Note that ovalbumin (QTAMVLVNAIV and NVMEERK) was identified in at least 5 slices of the gel. MW, molecular weight. PTMD, putative trans-membrane domain. Coverage, indicates the % of the protein sequence covered by the peptides identified.