Coatomer LPAAT-γ Merge

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Stanley Phillips
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1 DI: 1.138/n2273 Cotomr LPAAT-γ Mrg Tuuls (%) 1 5 < 5 nm 5~1nm 1~2nm 2~5nm >5nm 5~1nm < 5 nm 1~2nm 2~5nm >5nm 2 min 5 min 1 min 3 min < 5 nm 5~1nm 1~2nm 2~5nm >5nm < 5 nm 5~1nm 1~2nm 2~5nm >5nm Figur S1 Furthr hrtrizing LPAAT γ. () Cololiztion of pool of nognous LPAAT γ (r) with nognous otomr (grn) t th Golgi. Confol mirosopy ws prform on HL lls. Cotomr ws ll using n nti-otomr ntioy (CM1A1); r, 1 um. () Inhiition of LPAAT γ promots Golgi tuul formtion. Aftr th ition of n nti-lpaat-γ ntioy to th ronstitution systm, EM xmintion ws prform. Golgi tuuls within prtiulr lngths, s init, wr quntifi, n thn group n xprss s frtion of totl. Th mn from thr xprimnts with stnr rror is shown Mmilln Pulishrs Limit. All rights rsrv.

Stg I 1 μm + - + - P S P S P S P S β-cp ARF1 MAFP BEL Stg II 1 μm BEL MAFP - - + + + - ARFGAP1 P S P S P S P S β-cp Fig S2 Cotomr rls (% ontrol) 35 3 MAFP 2 15 BEL 1.1 1.

2 Stg I 1 μm P S P S P S P S β-cp ARF1 MAFP BEL Stg II 1 μm BEL MAFP ARFGAP1 P S P S P S P S β-cp Fig S2 Cotomr rls (% ontrol) 35 3 MAFP 2 15 BEL Inhiitor (μm) Cotomr PLA2-α Mrg No wsh.5 M KCl wsh 3. M KCl wsh PLA2α KDELR Cotomr Rls (% ontrol) M KCl Wsh 3.M KCl Wsh MAFP (μm) Figur S2 Furthr hrtrizing PLA2-α. () Efft of two PLA2 inhiitors on th CPI vsil ronstitution systm. Golgi mmrn ws wsh with.5m KCl to rtin nognous PLA2 tivity, n thn us in th two-stg ronstitution systm. Th first stg sssss ARF-pnnt rruitmnt of otomr onto Golgi mmrn. Th son stg sssss vsil formtion, whih is rflt y th rls of otomr from Golgi mmrn. () Dos-rspons nlysis of th two inhiitors on CPI vsil formtion. Th frtionl rls of β CP from Golgi mmrn ftr th son-stg inution ws normliz to ontrol inution (vhil only). Th mn from thr xprimnts with stnr rror is shown. () Cololiztion of pool of nognous PLA2 α (r) with nognous otomr (grn) t th Golgi. Confol mirosopy ws prform on HL lls. Cotomr ws ll using n nti-otomr ntioy (CM1A1); r: 1 μm. () Enognous PLA2 α is priphrl mmrn protin tht n rls y wshing Golgi mmrn mor stringntly. Isolt Golgi mmrn ws wsh s init n thn immunolott for protins s init. Immunolotting for th trnsmmrn KDELR rvls similr lvls of mmrn xmin. () Th ility of MAFP to promot CPI vsil formtion is rogt upon mor stringnt wshing of Golgi mmrn. Th CPI ronstitution systm ws prform using wsh Golgi mmrn n onntrtion of MAFP s init. Th son-stg inution ontin ARFGAP1 whn using.5m KCl-wsh Golgi mmrn n oth ARFGAP1 n BARS whn using 3M KCl-wsh Golgi mmrn. Th frtionl rls of β CP from Golgi mmrn ftr th son-stg inution ws normliz to ontrol inution (whih ontin vhil only). Th mn from thr xprimnts with stnr rror is shown Mmilln Pulishrs Limit. All rights rsrv.

3 4 ountsx1 SUPPLEMENTARY INFRMATIN ounts x 1 3 PC3H65 - m/z P3 - m/z P H PC3H55R - C16, m/z (R=C16H31) C18:1, m/z (R=C18H33) C18,m/z (R=C18H35) n[ ] C16:1,C18:1, C37H685 (637.54) C16:1,C18 / C16,C18:1, C37H75 (639.52) C14,C2 / C16,C18, C37H725 ( ) RC - C16, m/z C18:1,m/z C18, m/z [ ] n PC3H76R - C16, m/z C18:1,m/z C18, m/z C18:1, C39H725 ( ) C18,C18:1 / C16:1,C2, C39H745 ( ) m/z stnr ontrol CI 976 ounts x r x PA (C18, C18:1) PA (2C18:1) PA (C16, C18:1) m/z injt PA (C16, C18:1) (μg) Fig S3 Figur S3 Msuring lipi lvls on Golgi mmrn y mss sptromtry. () Frgmnttion pttrn of th PA spis xtrt from Golgi mmrn upon nlysis y LC-MS/MS, with frgmnts lult m/z for th rsptiv ftty i vrints init. () LC-MS/MS xprimnts on th iffrnt PA spis tt in Golgi mmrn xtrt. () LC-MS os rspons of PA stnr (C16, C18:1) s on r unr th urv of hromtogrms xtrt t m/z () Extrt mss sptr intifying DAG in Golgi mmrn xtrt. Arrows init th xpt m/z of formt ut [M+HC-]- of DAG spis. DAG spis wr ssign s on th mss ury (<1 ppm) of th tt ions ompr to lult m/z of formt uts init in prnthsis, n lso s on similrity of isotopi profil n rtntion tim with DAG stnr (C16, C18:1). () Extrt ion hromtogrms of DAG spis tt t m/z (C16,C18:1 or C16:1,C18) in iffrnt onitions r shown Mmilln Pulishrs Limit. All rights rsrv.

1 8 6 4 2 Cytosol (wt) Cyt (w/o CM) CI-976 PLA2α Tuuls (%) + - + - + - - + - + - + - - + + - - - - - - + + Vsils (pr msh) 15 12 9 6 3 Cytosol (wt) Cyt (w/o CM) CI-976 PLA2α + - + - + - - + - + - + -

5 8 6 4 2 Control NZ Tuuls (%) 1 5 < 5 nm 5~1nm 1~2nm 2~5nm >5nm 5~1nm < 5 nm 1~2nm 2~5nm >5nm 2 min 5 min 1 min 3 min < 5 nm 5~1nm 1~2nm 2~5nm >5nm < 5 nm 5~1nm 1~2nm 2~5nm >5nm Figur S4 Furthr

() Tuul formtion, ronstitut y inuting Golgi mmrn with ytosol, is inhiit upon th pltion of otomr from ytosol, n moult y opposing lipi nzymti tivitis.

4 Cytosol (wt) Cyt (w/o CM) CI-976 PLA2α Tuuls (%) Vsils (pr msh) Cytosol (wt) Cyt (w/o CM) CI-976 PLA2α Control Anti-CM Roun profils pr stk 1 P> Control NZ Tuuls (%) 1 5 < 5 nm 5~1nm 1~2nm 2~5nm >5nm 5~1nm < 5 nm 1~2nm 2~5nm >5nm 2 min 5 min 1 min 3 min < 5 nm 5~1nm 1~2nm 2~5nm >5nm < 5 nm 5~1nm 1~2nm 2~5nm >5nm Figur S4 Furthr hrtrizing th rltiv rols of CPI n lipi nzyms in vsil vrsus tuul formtion. () Tuul formtion, ronstitut y inuting Golgi mmrn with ytosol, is inhiit upon th pltion of otomr from ytosol, n moult y opposing lipi nzymti tivitis. In th iffrnt onitions s init, th lvl of tuuls (> 1 nm in lngth) ws quntifi y EM, n thn xprss s prntg of ll protrusions sn on Golgi mmrn. Th mn from thr xprimnts with stnr rror is shown. () Vsil formtion, ronstitut y inuting Golgi mmrn with ytosol, is inhiit y th pltion of otomr from ytosol, n moult y opposing lipi nzymti tivitis. In th iffrnt onitions s init, th lvl of vsils pr msh within th EM gri ws quntifi. Th mn from thr xprimnts with stnr rror is shown. () Efft of miroinjting n nti-otomr ntioy. Th Golgi ws visuliz y EM; r, 2 nm. In ontrol lls, Golgi stking n rion onntions r sn. In otomr inhiit lls, Golgi stking is prsrv, ut rion onntions r isrupt. () Noozol trtmnt os not signifintly ru th lvl of Golgi vsils. HL lls wr trt with onition s init (NZ, noozol). Th lvl of Golgi-ssoit vsils ws thn quntifi. Th mn from thr xprimnts with stnr rror is shown. Th stunt s t-tst ws prform for th two onitions, whih show no signifint iffrn (P >.5). () CPI tuul formtion still ours upon ut inhiition of PLD2. Th ronstitution systm ws prform. For th son stg inution, rominnt PLA2 α n nti-pld2 ntioy wr itionlly. Golgi tuuls within prtiulr lngths, s init, wr quntifi, n thn group n xprss s frtion of totl. Th mn from thr xprimnts with stnr rror is shown Mmilln Pulishrs Limit. All rights rsrv.

Cotomr rls (%) Tuuls (%) 1 5 1 5 Non PLD2 inhi PLD1 inhi >5nm 2~5nm 1~2nm 5~1nm < 5 nm >5nm 2~5nm 1~2nm 5~1nm < 5 nm >5nm 2~5nm 1~2nm 5~1nm < 5 nm >5nm 2~5nm 1~2nm 5~1nm < 5 nm 2 min 5 min 1 min 3

5 Cotomr rls (%) Tuuls (%) Non PLD2 inhi PLD1 inhi >5nm 2~5nm 1~2nm 5~1nm < 5 nm >5nm 2~5nm 1~2nm 5~1nm < 5 nm >5nm 2~5nm 1~2nm 5~1nm < 5 nm >5nm 2~5nm 1~2nm 5~1nm < 5 nm 2 min 5 min 1 min 3 min VSVG-KDELR ololizing with gintin (%) 1 Prmissiv Non-prmissiv 5 VSVG ololizing with TGN46 (%) 5 Non PLD2 inhi PLD1 inhi 1 Control PLD2 inhi min 12 min Figur S5 Phrmologi inhiition of PLD2 inhiits rtrogr CPI vsiulr, ut not ntrogr CPI tuulr, trnsport. () PLD2 tivity is rquir for CPI vsil formtion. Inhiitors tht trgt ithr PLD1 or PLD2 (s mtho stion for ompoun nm) wr to th ronstitution systm. Th frtion of otomr rls ftr th son-stg inution ws thn quntifi. Th mn with stnr rror from thr xprimnts is shown. () Th PLD2 inhiitor inus th umultion of us on Golgi mmrn with onstrit nks. Golgi mmrn in th ov xprimnt tht us th PLD2 inhiitor ws xmin y EM; r, 5 nm. () Rtrogr trnsport of VSVG-KDELR is inhiit y th ition of th PLD2, ut not th PLD1, inhiitor. HL lls wr trt with inhiitors s init, follow y quntittion of CPI-pnnt rtrogr trnsport s rflt y th gr ololiztion twn VSVG-KDELR n Golgi mrkr (gintin) t th nonprmissiv tmprtur. Th mn with stnr rror from thr xprimnts is shown. () Th PLD2 inhiitor os not fft CPI tuul formtion. Th PLD2 inhiitor ws to th ronstitution systm tht us rominnt PLA2 α to inu CPI tuul formtion. Golgi tuuls within prtiulr lngths, s init, wr quntifi, n thn group n xprss s frtion of totl. Th mn from thr xprimnts with stnr rror is shown. () Antrogr trnsport through th Golgi stks is not fft y th ition of th PLD2 inhiitor. VSVG-ts45 ws xprss in HL lls, n thn llow to umult t th pr-golgi omprtmnt y shifting ll inution from 4 o C to 15 o C. Clls wr thn trt with th PLD2 inhiitor, n thn shift to 2 o C for tims init. Cololiztion of VSVG-ts45 with TGN46 ws susquntly quntifi. Th mn from thr xprimnts with stnr rror is shown Mmilln Pulishrs Limit. All rights rsrv.

6 Fig 2C Fig 3C Fig 5G Figur S6 Full gl sns for figurs s init Mmilln Pulishrs Limit. All rights rsrv.

mch log height Counts GFP log height mch log height Counts GFP log height mch log height Counts high flux % Hist: 8.34 EBSS shctrl Counts

mch log height Counts GFP log height mch log height Counts GFP log height mch log height Counts high flux % Hist: 8.34 EBSS shctrl Counts DOI:.8/n2886 mchrry-gfp-lc3 Autophgy Rportr: Autophgosom Autolysosom mchrry GFP LC3 mchrry GFP LC3 X ph >6. ph