Predicted membrane topologies of members of the TMBIM family. N and C terminally tagged alleles localise at the Golgi.
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1 Supplementary fig. 1. Predicted membrane topologies of members of the TMBIM family. Multiple amino acid sequence alignment of members of the TMBIM family generated with Clustal W, with transmembrane regions I VII highlighted in yellow as predicted by MEMSAT-VSM. Transmembrane regions which are not predicted to be present by some bioinformatics tool such as MEMSAT3 are highlighted in gray. Supplementary fig. 2. N and C terminally tagged alleles localise at the Golgi. Confocal microscopy of HeLa cells transfected with vgaap and hgaap C terminally tagged with HA or YFP, and HeLa cell line expressing hgaap tagged with V5 at the N terminus. Cells were fixed, permeabilised with triton X-100, and probed with the Golgi marker, GM130, as well as HA or V5 antibody. Scale bar, 20 µm. Supplementary fig. 3. HA tags individually inserted between vgaap TMDs alters the localisation of some alleles to the ER. HeLa cells transfected with vgaap alleles containing a HA tag between TM regions 1-2, 2-3, 3-4, 4-5, 5-6, and 6-7. Cells were fixed, permeabilised with triton and stained with Golgi and ER markers, GM 130 and PDI antibody respectively. Supplementary fig. 4. The ER localisation of BI-1 is not altered by fusion of fluorescent proteins or the insertion of a HA tag within BI-1. HeLa cells were transfected with BI-1 fused to CFP at the N or C terminus, and BI-1-CFP with a HA tag inserted between TM 6-7. Cells were fixed and permeabilised, followed by immunostaining with anti-pdi, an ER marker. Supplementary fig. 5. Expression of vgaap-ha by transient transfection is similar to endogenous levels of vgaap during infection. HeLa cells were mock infected, infected with the Evans strain of VACV in which vgaap has been deleted ( vgaap), or the vgaap VACV Evans in which vgaap-ha was inserted into its original locus (vgaap-ha Rev), or transfected with vgaap-ha plasmid. The lysate was immunoblotted with anti-ha, anti-d8 as an infection control and anti-tubulin as a loading control. Supplementary fig. 6. Digitonin treatment is a reliable technique for selectively permeabilising the plasma membrane for antibody mediated topology assays. The efficiency of digitonin treatment in topology assays was addressed in cells expressing C terminal fluorescently tagged vgaap, hgaap and BI-1 alleles; the N terminal V5-tagged hgaap cell line; two vgaap HA insertions of determined opposite orientations between TM 1-2 and 4-5; and the BI-1 HA insertion between TM 6-7. The mean percentage of cells positive (black) or negative (gray) for GFP antibody staining within the transfected cell population, were counted by confocal microscopy. The transfected cell population is marked by the expression of the fluorescent tag for all alleles except the V5-hGAAP cell line where the whole cell population was used. Each allele was quantified in duplicate experiments, apart from BI-1 alleles which were derived from a single experiment.
2 Supplementary fig. 1. MNIF-DRKI-NF-DAL MTQG-KLSV--ANKAPGT----EGQQQVHGEK---KEAPAVPSA MSHE-KSFLVSGDNYPPPNPGYPGGPQPPMPP---YAQPPYPGAP----- MLAV-RLVC--LRTLPSR----VFHP------AFTKASPVVKNSITKNQW APEDLQHSE--ARNAPSG----TAQVPAAGVPPQPVSSHVQPQP MADP DPR L PPSYEEATSG-EG MKAGAFPPAP TAVPL YPQPPFQPSPYG-QPGYP------HGPSPYPQGGYPQGPYPQGGYPQGP- LLTPSREYATKTRIGIRRGRTGQELKEAALEPSM EKMFK --PPPYEDRN----P LYPGPLPPGGYGQPSVLPGGYPAYPG -----YPRSS IEDDFN K FSHIT---P----- HPSWAYVDPSS-SSSYDNGF YPQEGYPQGPYPQGGYPQGPYPQSPFPPNPYGQPQVFPGQDPDSPQHG-N IDQMGRWF---V-AGG A-AVGLGALCY YPQPGYGHP----AGYPQPMPPTHPMPMN YGPGHG STQQHLKKVYASFALCMFVA-AAG PT--GDHELFTTFSWDDQKVRRVFVRKVYTILLIQLLVT-LAVVA YQEEGPPSYYDNQDFPATNWDDKSIRQAFIRKVFLVLTLQLSVT-LSTVS YGLGLSN--EIGAIEKAVIWPRYV-K-DRIHSTYMYLAGSIGLTALSAIA YD-GEER--AVSDSFGPGEWDDRKVRHTFIRKVYSIISVQLLIT-VAIIA YG SSVASATVHIRMAFLRKVYSILSLQVLLT-TVTST ::..: : : :: --AYVHMVTHFIQAGL-LSALGSLI------LMIWLMATPHSHETEQKRL LFTFCDPVKDYVQANP-GWYWASYAVFFATYLTLACCSGPRR--HFPWNL VFTFVAEVKGFVRENV-WTYYVSYAVFFISLIVLSCCGDFRR--KHPWNL IS-RTPVLMNFMMRGSWVTIGVTFAAMVGAGMLVRSIPYDQS--PGPKHL IFTFVEPVSAFVRRNV-AVYYVSYAVFVVTYLILACCQGPRR--RFPWNI VFLYFESVRTFVHESP-ALILL-FALGS-LGLIF-ALILNRH--KYPLNL :. :.: GLLAGFAFLTGVGLGPALEFCIAVNPSILPTAFMGTAMIFTCFTLSALYA ILLTVFTLSMAYLTGMLSSY---YNTTSVLLCLGITALVCLSVTV-FSFQ VALSVLTASLSYMVGMIASF---YNTEAVIMAVGITTAVCFTVVI-FSMQ AWLLHS-GVMGAVVAPL-TI---LGGPLLIRAAWYTAGIVGGLSTVAMCA ILLTLFTFAMGFMTGTISSM---YQTKAVIIAMIITAVVSISVTI-FCFQ YLLFGFTLLEALTVAVVVTF---YDVYIILQAFILTTTVFFGLTV-YTLQ *.. :. *: :. RRRSYLFLGGILMSALSLLLLSS----LGNVFFG-----SIWLFQANLYV TKFDFTSCQGVLFVLLMTLFFSGLILAILLPFQY-----VPWLHAVYAAL TRYDFTSCMGVLLVSMVVLFIFA----ILCIFIR-----NRILEIVYASL PSEKFLNMGAPLGVGLGLVFVSS----LGSMFLPPTTVAGATLYSVAMYG TKVDFTSCTGLFCVLGIVLLVTGIVTSIVLYFQY-----VYWLHMLYAAL SKKDFSKFGAGLFALLWILCLSG----FLKFFFY-----SEIMELVLAAA.:. : :.. : * : VI III V GLVVMCGFVLFDTQLIIEKAEH--GD------QDYIWHCIDLFLDFITVF GAGVFTLFLALDTQLLMGNRRHSLSP------EEYIFGALNIYLDIIYIF GALLFTCFLAVDTQLLLGNKQLSLSP------EEYVFAALNLYTDIINIF GLVLFSMFLLYDTQKVIKRA--EVSPMYGVQKYDPINSMLSIYMDTLNIF 1 II IV I VII
3 GAICFTLFLAYDTQLVLGNRKHTISP------EDYITGALQIYTDIIYIF GALLFCGFIIYDTHSLMH----KLSP------EEYVLAAISLYLDIINLF * : *: **: ::. : : :.:: * : :* RKLMMILAMNEKDKKKEKK TFFLQLF GTNRE * identity LYILTII GRAKE : high similarity MRVATMLATG----GNRKK. low similarity TFVLQLM G-DRN LHLLRFL---E---AVNKK. :: :: 2
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33VASTVNGATSANNHGEPPS51PADARPR58
Pro-rich region Trans-membrane region 214 246 359 381 UL50 1 397 211SSRTAS216PPPPPR222 NLS CR1 CR2 CR3 CR4 UL53 1 376 11RERRS15ALRS19LLRKRRR25 33VASTVNGATSANNHGEPPS51PADARPR58 FIG S1. UL97 phosphorylation
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