MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface. A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface. EMBO J. (0261-4189) 2012 imex curation Wakana Y., van Galen J., Meissner F., Scarpa M., Polishchuk RS., Mann M., Malhotra V. Only protein-protein interactions human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 fluorescence imaging fluorescence microscopy fluorescence imaging experimental particp experimental participant identification experimental particp Wakana Y., van Galen J., Meissner F., Scarpa M., Polishchuk RS., Mann M., Malhotra V. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation rab8a_human Ras-related protein Rab-8A Oncogene c-mel RAB8A MEL RAB8 protein protein human Homo sapiens Human MAKTYDYLFKLLLIGDSGVGKTCVLFRFSEDAFNSTFISTIGIDFKIRTIELDGKRIKLQIWDTAGQERFRTITTAYYRGAMGIMLVYDITNEKSFDNIRNWIRNIEEHASADVEKMILGNKCDVNDKRQVSKERGEKLALDYGIKFMETSAKANINVENAFFTLARDIKAKMDKKLEGNSPQGSNQGVKITPDQQKRSSFFRCVLL DIP protein P61006 original sequence version: 82 DIP protein P61006 original sequence version: 100 DIP protein P61006 original sequence version: 128 DIP protein P61006 original sequence version: 132 AA52DBF54A2CD056 syt2_human Synaptotagmin-2 SYT2 Synaptotagmin II protein protein human Homo sapiens Human MRNIFKRNQEPIVAPATTTATMPIGPVDNSTESGGAGESQEDMFAKLKEKLFNEINKIPLPPWALIAIAVVAGLLLLTCCFCICKKCCCKKKKNKKEKGKGMKNAMNMKDMKGGQDDDDAETGLTEGEGEGEEEKEPENLGKLQFSLDYDFQANQLTVGVLQAAELPALDMGGTSDPYVKVFLLPDKKKKYETKVHRKTLNPAFNETFTFKVPYQELGGKTLVMAIYDFDRFSKHDIIGEVKVPMNTVDLGQPIEEWRDLQGGEKEEPEKLGDICTSLRYVPTAGKLTVCILEAKNLKKMDVGGLSDPYVKIHLMQNGKRLKKKKTTVKKKTLNPYFNESFSFEIPFEQIQKVQVVVTVLDYDKLGKNEAIGKIFVGSNATGTELRHWSDMLANPRRPIAQWHSLKPEEEVDALLGKNK mint homomint May have a regulatory role in the membrane interactions during trafficking of synaptic vesicles at the active zone of the synapse. It binds acidic phospholipids with a specificity that requires the presence of both an acidic head group and a diacyl backbone BE3855E9CDE2D76E myh9_human Myosin-9 MYH9 Myosin heavy chain 9 Myosin heavy chain, non-muscle IIa Non-muscle myosin heavy chain IIa Cellular myosin heavy chain, type A Non-muscle myosin heavy chain A protein protein human Homo sapiens Human MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVENGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVVINPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGKTENTKKVIQYLAYVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIRINFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKYRFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNTDQASMPDNTAAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEALAKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQQLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKATDKSFVEKVMQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIATLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKLMATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEFRQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDLKITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPLLQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCAEAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEESARQKLQLEKVTTEAKLKKLEEEQIILEDQNCKLAKEKKLLEDRIAEFTTNLTEEEEKSKSLAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQLAKKEEELQAALARVEEEAAQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDLGEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEAKTHEAQIQEMRQKHSQAVEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQELQVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDFSALESQLQDTQELLQEENRQKLSLSTKLKQVEDEKNSFREQLEEEEEAKHNLEKQIATLHAQVADMKKKMEDSVGCLETAEEVKRKLQKDLEGLSQRHEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSACNLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELERLNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLRLEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAVAARKKLEMDLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKSMEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEELEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEMEGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRTEKKLKDVLLQVDDERRNAEQYKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSLKNKLRRGDLPFVVPRRMARKGAGDGSDEEVDGKADGAEAKPAE DIP protein P35579 original sequence version: 126 DIP protein P35579-1 original sequence version: 142 DIP protein P35579-1 original sequence version: 133 DIP protein P35579 original sequence version: 186 DIP protein P35579-1 original sequence version: 186 DIP protein P35579-1 original sequence version: 146 DIP protein P35579 original sequence version: 146 588F84BB8C106E6F glyco_vsivo Glycoprotein G protein protein vsivo Vesicular stomatitis Indiana virus (strain Orsay) MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAAIVQVTPHHVLVDEYTGEWVDSQFINGKCSNDICPTVHNSTTWHSDYKVKGLCDSNLISTDITFFSEDGELSSLGKEGTGFRSNYFAYETGDKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPVFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSLGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDETLFFGDTGLSKNPIEFVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIYLCIKLKHTKKRQIYTDIEMNRLGK A10E67B6F8138179 lysc_human Lysozyme C LYZ LZM 1,4-beta-N-acetylmuramidase C protein protein human Homo sapiens Human MKALIVLGLVLLSVTVQGKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV 8ECFD276BEB2678A c3ptt6_human PAUF protein protein human Homo sapiens Human MWRVPGTTRRPVTGESPGMHRPEAMLLLLTLALLGGPTWAGKMYGPGGGKYFSTTEDYDHEITGLRVSVGLLLVKSVQVKLGDSWDVKLGALGGNTQEVTLQPGEYITKVFVAFQAFLRGMVMYTSKDRYFYFGKLDGQISSAYPSQEGQVLVGIYGQYQLLGIKSIGFEWNYPLEEPTTEPPVNLTYSANSPVGR 090C9C973F510E5A rab6a_human Ras-related protein Rab-6A RAB6A RAB6 protein protein human Homo sapiens Human MSTGGDFGNPLRKFKLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTVRLQLWDTAGQERFRSLIPSYIRDSTVAVVVYDITNVNSFQQTTKWIDDVRTERGSDVIIMLVGNKTDLADKRQVSIEEGERKAKELNVMFIETSAKAGYNVKQLFRRVAAALPGMESTQDRSREDMIDIKLEKPQEQPVSEGGCSC C9A3C2DDBF6C3E9E tgon2_human Trans-Golgi network integral membrane protein 2 TGOLN2 TGN46 TGN51 Trans-Golgi network glycoprotein 46 Trans-Golgi network glycoprotein 48 Trans-Golgi network glycoprotein 51 Trans-Golgi network protein 2 protein protein human Homo sapiens Human MRFVVALVLLNVAAAGAVPLLATESVKQEEAGVRPSAGNVSTHPSLSQRPGGSTKSHPEPQTPKDSPSKSSAEAQTPEDTPNKSGAEAKTQKDSSNKSGAEAKTQKGSTSKSGSEAQTTKDSTSKSHPELQTPKDSTGKSGAEAQTPEDSPNRSGAEAKTQKDSPSKSGSEAQTTKDVPNKSGADGQTPKDGSSKSGAEDQTPKDVPNKSGAEKQTPKDGSNKSGAEEQGPIDGPSKSGAEEQTSKDSPNKVVPEQPSRKDHSKPISNPSDNKELPKADTNQLADKGKLSPHAFKTESGEETDLISPPQEEVKSSEPTEDVEPKEAEDDDTGPEEGSPPKEEKEKMSGSASSENREGTLSDSTGSEKDDLYPNGSGNGSAESSHFFAYLVTAAILVAVLYIAHHNKRKIIAFVLEGKRSKVTRRPKASDYQRLDQKS 6F72467FED83A1A3 kpcd2_human Serine/threonine-protein kinase D2 PKD2 nPKC-D2 HSPC187 PRKD2 protein protein human Homo sapiens Human MATAPSYPAGLPGSPGPGSPPPPGGLELQSPPPLLPQIPAPGSGVSFHIQIGLTREFVLLPAASELAHVKQLACSIVDQKFPECGFYGLYDKILLFKHDPTSANLLQLVRSSGDIQEGDLVEVVLSASATFEDFQIRPHALTVHSYRAPAFCDHCGEMLFGLVRQGLKCDGCGLNYHKRCAFSIPNNCSGARKRRLSSTSLASGHSVRLGTSESLPCTAEELSRSTTELLPRRPPSSSSSSSASSYTGRPIELDKMLLSKVKVPHTFLIHSYTRPTVCQACKKLLKGLFRQGLQCKDCKFNCHKRCATRVPNDCLGEALINGDVPMEEATDFSEADKSALMDESEDSGVIPGSHSENALHASEEEEGEGGKAQSSLGYIPLMRVVQSVRHTTRKSSTTLREGWVVHYSNKDTLRKRHYWRLDCKCITLFQNNTTNRYYKEIPLSEILTVESAQNFSLVPPGTNPHCFEIVTANATYFVGEMPGGTPGGPSGQGAEAARGWETAIRQALMPVILQDAPSAPGHAPHRQASLSISVSNSQIQENVDIATVYQIFPDEVLGSGQFGVVYGGKHRKTGRDVAVKVIDKLRFPTKQESQLRNEVAILQSLRHPGIVNLECMFETPEKVFVVMEKLHGDMLEMILSSEKGRLPERLTKFLITQILVALRHLHFKNIVHCDLKPENVLLASADPFPQVKLCDFGFARIIGEKSFRRSVVGTPAYLAPEVLLNQGYNRSLDMWSVGVIMYVSLSGTFPFNEDEDINDQIQNAAFMYPASPWSHISAGAIDLINNLLQVKMRKRYSVDKSLSHPWLQEYQTWLDLRELEGKMGERYITHESDDARWEQFAAEHPLPGSGLPTDRDLGGACPPQDHDMQGLAERISVL 8BAB45E8F99D23E7 lyz-pauf 1 Lysozyme C 6 immunostaining immunostaining experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined PAUF 7 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization f3f homomint mint We tested whether the secretory cargo, lysozyme C, was contained in the CARTS. Lysozyme C-3 FLAG was co-expressed with PAUF-MycHis in HeLa cells. In all, 93% (n¼613 punctate elements in 5 cells) of PAUF containing CARTS co-localized with lysozyme C-3 FLAG (Figure 3F). syt2-tgoln2 1 SYT2 3 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined TGN46 9 immunostaining immunostaining experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell colocalization colocalization f5b homomint mint Syt II-3 FLAG was expressed in HeLa cells and visualized by fluorescence microscopy. Syt II was found to co-localize with TGN46 (Figure 5B, top row). pauf-syt2-1 1 SYT2 3 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined PAUF 7 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization f5b homomint mint We next tested the localization of PAUF-MycHis and Syt II-3 FLAG in cells co-expressing these proteins. Syt II-3 FLAG was localized to the plasma membrane, the TGN, and a cluster of punctate elements (Figure 5B, bottom row). pauf-pauf 1 PAUF 7 tag fluorescence tag visualisation by fluorescence tag fluorescence experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell rfp tag rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined PAUF 7 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization f9a homomint mint As a control, PAUF-mRFP was co-expressed with PAUF-MycHis in HeLa cells and incubated as described above. PAUF-mRFP and PAUF-MycHis revealed 498% co-localization (n¼827 punctate elements in 10 cells) (Figure 9A). pauf-prkd2 1 PKD2-KD 10 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined PAUF 7 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization f3b homomint mint HeLa cells were co-transfected with plasmids for glutathione S transferase (GST)-PKD2-KD and PAUFMycHis. The cells were visualized with anti-GST and anti- Myc antibodies to localize GST-PKD2-KD and PAUF-MycHis, respectively. They were found to co-localize at the Golgi apparatus (Figure 3B). prkd2-syt2 1 SYT2 3 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined PKD2-KD 10 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization f5b homomint mint In cells coexpressing Syt II-3 FLAG and GST-PKD2-KD, they were found to co-localize at the TGN and the attached tubules (Figure 5B, middle row). myh9-prkd2 1 Myosin IIa 4 tag fluorescence tag visualisation by fluorescence tag fluorescence experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined PKD2-KD 10 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization f7a homomint mint GFP-myosin IIa was found in the perinuclear region and many of the GFP-myosin IIa containing punctae co-localized with GST-PKD2-KD (Figure 7A). pauf-rab6a 1 Rab6a 8 tag fluorescence tag visualisation by fluorescence tag fluorescence experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined PAUF 7 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization homomint mint We tested whether Rab proteins reported to be involved in post Golgi trafficking were contained in the CARTS. Rab6a-green fluorescent protein (GFP), Rab8a-GFP, and Rab11a-GFP were co-expressed with PAUF-MycHis in HeLa cells and the cells were visualized by fluorescence microscopy (Figure 6A). An anti-Myc antibody was used to identify PAUF containing CARTS and the results revealed the co-localization with both Rab6a and Rab8a, but not with Rab11a (Figure 6A and B). f6a f6b pauf-rab8a 1 Rab8a 2 tag fluorescence tag visualisation by fluorescence tag fluorescence experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined PAUF 7 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization homomint mint We tested whether Rab proteins reported to be involved in post Golgi trafficking were contained in the CARTS. Rab6a-green fluorescent protein (GFP), Rab8a-GFP, and Rab11a-GFP were co-expressed with PAUF-MycHis in HeLa cells and the cells were visualized by fluorescence microscopy (Figure 6A). An anti-Myc antibody was used to identify PAUF containing CARTS and the results revealed the co-localization with both Rab6a and Rab8a, but not with Rab11a (Figure 6A and B). f6a f6b pauf-rab6a-1 1 Rab6a 8 immunostaining immunostaining experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell PAUF 7 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization homomint sf3 mint g-pauf-1 1 VSV-G 5 tag fluorescence tag visualisation by fluorescence tag fluorescence experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined PAUF 7 anti tag immunost anti tag immunostaining anti tag immunost experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell tandem tag tandem tag tandem tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization homomint mint HeLa cells co-transfected with plasmids for PAUF-MycHis and VSV-G-GFP were incubated for 18 h at 401C. The cells were shifted to 201C for 2 h in the presence of cycloheximide and then after 0, 5, 15, or 60 min at 321C fixed and stained with anti-Myc antibody for fluorescence microscopy (Figure 9B; Supplementary Figure S7B). At the earliest time point (0 min), PAUF-MycHis, VSV-G-GFP, and TGN46 clearlyco-localized at the perinuclear region (Supplementary Figure S7B). A small number of PAUF containing carriers was evident even at the earliest time point (0 min). Probably, these carriers were generated during the 401C incubation, after which their fusion with the plasma membrane was delayed by the incubation at 201C. After 15 min at 321C, there was a significant increase in the number of VSV-GGFP and PAUF-MycHis containing carriers (Figure 9B; Supplementary Figure S7B). We found that o20% (n¼1120 punctate elements in 9 cells) of the PAUF-MycHis containing CARTS contained VSV-G-GFP. Similarly, o20% (n¼1073 punctate elements in 9 cells) of VSV-G-GFP containing carriers contained PAUF-MycHis. At 60 min, VSV-G-GFP was detected at the plasma membrane (Supplementary Figure S7). Interestingly, a number of these VSV-G-GFP and PAUF-MycHis containing carriers were located in close vicinity (Figure 9B; Supplementary Figure S7). They might be formed at the same exit site of the TGN and use the same track leading to the plasma membrane. f9b sf7b