IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. J. Biol. Chem. (0021-9258) 1999 imex curation Jungmann J., Rayner JC., Munro S. sean@mrc-lmb.cam.ac.uk jyoti Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Jungmann J., Rayner JC., Munro S. sean@mrc-lmb.cam.ac.uk Accepted 2010-MAY-21 by JYOTI. J. Biol. Chem. (0021-9258) 1999 jyoti Only protein-protein interactions imex curation The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. J. Biol. Chem. (0021-9258) 1999 imex curation Jungmann J., Rayner JC., Munro S. sean@mrc-lmb.cam.ac.uk jyoti Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Jungmann J., Rayner JC., Munro S. sean@mrc-lmb.cam.ac.uk Accepted 2010-MAY-21 by JYOTI. J. Biol. Chem. (0021-9258) 1999 jyoti Only protein-protein interactions imex curation The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. J. Biol. Chem. (0021-9258) 1999 imex curation Jungmann J., Rayner JC., Munro S. sean@mrc-lmb.cam.ac.uk jyoti Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column western blot western blot Immuno blot Immuno blot Jungmann J., Rayner JC., Munro S. Sephacryl S400 gel filtration column sean@mrc-lmb.cam.ac.uk Accepted 2010-MAY-21 by JYOTI. J. Biol. Chem. (0021-9258) 1999 jyoti Only protein-protein interactions imex curation mnn10_yeast Probable alpha-1,6-mannosyltransferase MNN10 MNN10 BED1 YDR245W YD8419.12 Mannan polymerase II complex MNN10 subunit Bud emergence delay protein 1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSVPYNSQLPISNHLEYDEDEKKSRGSKLGLKYKMIYWRKTLCSSLARWRKLILLISLALFLFIWISDSTISRNPSTTSFQGQNSNDNKLSNTGSSINSKRYVPPYSKRSRWSFWNQDPRIVIILAANEGGGVLRWKNEQEWAIEGISIENKKAYAKRHGYALTIKDLTTSKRYSHEYREGWQKVDILRQTFREFPNAEWFWWLDLDTMIMEPSKSLEEHIFDRLETLADRELKSFNPLNLRDDIPYVDYSEEMEFLITQDCGGFNLGSFLIKNSEWSKLLLDMWWDPVLYEQKHMVWEHREQDALEALYENEPWIRSRIGFLPLRTINAFPPGACSEYSGDSRYFYSEKDHDFVVNMAGCNFGRDCWGEMQYYTTLMEKLNRKWYTRFFFP FAF7DBE3122ECC8E mnn9_yeast Mannan polymerase complexes subunit MNN9 MNN9 YPL050C P7102.01 M-Pol II subunit MNN9 protein protein yeast Saccharomyces cerevisiae Baker's yeast MSLSLVSYRLRKNPWVNIFLPVLAIFLIYIIFFQRDQSLLGLNGQSISQHKWAHEKENTFYFPFTKKYKMPKYSYKKKSGWLFNDHVEDIIPEGHIAHYDLNKLHSTSEAAVNKEHILILTPMQTFHQQYWDNLLQLNYPRELIELGFITPRTATGDLALKKLENAIKKVQTDKKTQRFSKITILRQNSQSFDKLMEKERHALDVQKERRAAMALARNELLFSTIGPHTSWVLWLDADIIETPPSLIQDMTKHNKAILAANIYQRFYDEEKKQPSIRPYDFNNWQESDTGLEIASQMGDDEIIVEGYAEIATYRPLMAHFYDANGVPGEEMALDGVGGGCTLVKAEVHRDGAMFPNFPFYHLIETEGFAKMAKRLNYDVFGLPNYLVYHIEEENH 461783BB4D700987 461783BB4D700987 mnn11_yeast Probable alpha-1,6-mannosyltransferase MNN11 MNN11 YJL183W J0425 Mannan polymerase II complex MNN11 subunit protein protein yeast Saccharomyces cerevisiae Baker's yeast MAIKPRTKGKTYSSRSVGSQWFNRLGFKQNKYGTCKFLSIITAFVFILYFFSNRFYPISRSAGASYSPSHGLYINEIPASSRLIYPHVEHVPVLKQMTVRGLYITRLEVDGSKRLILKPEENALTDEEKKKTTDQILLVKHSFLDHGKLVYRKSNDAPEVVVVTLIDFENYELETIIQIVQNRVDYAQKHQYGVYIRWIQEFLPVLENQNLAESYEFIKPLVIRAAMHAFPTAKYIHFVDQDALLMNLDLSLQKYLLDPKIMDLALLKNVPVVANSNIKTYNHFEYSSAKIIIPHDADGNIDASSFVIANDFYGKALIDYLNDPLLRNFPWDNTGDKLSAAIGHILQWHPTLLGKTAIVIPKVLASQYDASLDQEGESGNGASNGDVYHYNEGDLAASFKGCRSRGTCASEIGHMYQKIKKS B1764ADBC19F9E58 anp1_yeast Mannan polymerase II complex ANP1 subunit Aminonitrophenyl propanediol resistance protein ANP1 GEM3 YEL036C SYGP-ORF28 protein protein yeast Saccharomyces cerevisiae Baker's yeast MKYNNRKLSFNPTTVSIAGTLLTVFFLTRLVLSFFSISLFQLVTFQGIFKPYVPDFKNTPSVEFYDLRNYQGNKDGWQQGDRILFCVPLRDASEHLPMFFNHLNTMTYPHNLIDLSFLVSDSSDNTMGVLLSNLQMAQSQQDKSKRFGNIEIYEKDFGQIIGQSFSDRHGFGAQGPRRKLMARARNWLGSVALKPYHSWVYWRDVDVETIPTTIMEDLMHHDKDVIVPNVWRPLPDWLGNIQPYDLNSWKESEGGLQLADSLDEDAVIVEGYPEYATWRPHLAYMRDPNGNPEDEMELDGIGGVSILAKAKVFRTGSHFPAFSFEKHAETEAFGRLSRRMNYNVIGLPHYVIWHIYEPSSDDLKHMAWMAEEEKRKLEEERIREFYNKIWEIGFEDVRDQWNEERDSILKNIDSTLNNKVTVDWSEEGDGSELVDSKGDFVSPNNQQQQQQQQQQQQQQQQQQQQQQLDGNPQGKPLDDNDKNKKKHPKEVPLDFDPDRN 845B395CE548CD14 845B395CE548CD14 hoc1_yeast Putative glycosyltransferase HOC1 HOC1 YJR075W J1830 Mannan polymerase II complex HOC1 subunit M-Pol II subunit Hoc1p protein protein yeast Saccharomyces cerevisiae Baker's yeast MAKTTKRASSFRRLMIFAIIALISLAFGVRYLFHNSNATDLQKILQNLPKEISQSINSANNIQSSDSDLVQHFESLAQEIRHQQEVQAKQFDKQRKILEKKIQDLKQTPPEATLRERIAMTFPYDSHVKFPAFIWQTWSNDEGPERVQDIKGMWESKNPGFAHEVLNHDVINALVHHYFYSIPEILETYEALPSIILKIDFFKYLILLVHGGVYADIDTFPVQPIPNWIPEELSPSDIGLIVGVEEDAQRADWRTKYIRRLQFGTWIIQAKPGHPVLREIISRIIETTLQRKRDDQLNVNLRNDLNIMSWTGSGLWTDTIFTYFNDFMRSGVREKVTWKLFHNLNQPKLLSDVLVFPKFSFNCPNQIDNDDPHKKFYFITHLASQFWKNTPKVEQK 8BF77385510DE6B0 8BF77385510DE6B0 mnn10-anp1-1 1 7 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association Fig. 1B Moreover, blotting of the supernatant following precipitation revealed that the Anp1p was quantitatively precipitated by the tagged Mnn10p, indicating that all of the Anp1p is associated with complexes that contain Mnn10p (Fig. 1 B). mnn10-mnn11-1 2 4 unspecified role unspecified role bait bait c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 6 unspecified role unspecified role prey prey c-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association Fig. 2A A, immunoblots of proteins isolated by immunoprecipitation from strains expressing Mnn10p and Mnn11p tagged with either the Myc or HA epitopes as indicated. mnn10-mnn11-2 2 6 unspecified role unspecified role bait bait c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 4 unspecified role unspecified role prey prey c-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association Fig. 2A A, immunoblots of proteins isolated by immunoprecipitation from strains expressing Mnn10p and Mnn11p tagged with either the Myc or HA epitopes as indicated. mnn10-hoc1-1 2 4 unspecified role unspecified role bait bait c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 8 unspecified role unspecified role prey prey c-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association Fig. 2B mnn11-hoc1-1 2 6 unspecified role unspecified role bait bait c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 8 unspecified role unspecified role prey prey c-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association Fig. 2B mnn9-anp1-4 3 5 unspecified role unspecified role neutral component neutral component c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 7 unspecified role unspecified role neutral component neutral component physical association physical association The immunoprecipitates were then probed with specific antisera, and Figure 3 shows that both Anp1p and Van1p co-precipitated with Mnn9p. Fig. 4B When a whole cell lysate was applied to the same column, Anp1p elutes in a similar position (Fig.4 B) and an epitope-tagged version of Mnn9p elutes in two peaks corresponding to the positions of the isolated M-Pol I and M-Pol II.