ユビキチンリガーゼRFFLによるエンドソーム機能制御機構の解明

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ユビキチンリガーゼRFFLによるエンドソーム機能制

御機構の解明

著者

酒井了平

学位名

博士（理学）

学位授与機関

関西学院大学

学位授与番号

34504甲第692号

URL

http://hdl.handle.net/10236/00028258

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ʬɚħʰġħħ¯ɰƈ

qPcO_

RFFL .=@

Kh`|ƱȻÖŘƱƭ/ɠƑ

˖˔˕˗

ʬɚħʰġħġħʰǢľħǾȐȉ

ǤăȉħĵƄ

ʝ Ń

(3)

Ǭ

Ǭɭɓ

ƚɰƈ*0§/ǬɭD¶ǥ@˓

CME: clathrin-mediated endocytosisːR\¸ĦǵKhXHgZ\ˑ CIE: clathrin-independent endocytosisːR\ʽ¸ĦǵKhXHgZ\ˑ EE: early endosomeːÓƗKh`|ˑ

LE: late endosomeːŔƗKh`|ˑ LY: lysosomesː``|ˑ

TGN: trans-Golgi networkːg\W[kdgRˑ RE: recycling endosomeːXHRSKh`|ˑ PM: plasma membraneːŎɸȿˑ

ERC: endocytic recycling compartment MVBs: multivesicular bodiesːĠȺ²ˑ ILVs: intraluminal vesiclesːȽËȿķȺˑ

EHDs: eps15 homology domain containing proteins Rab11-FIPs: Rab11-family interacting proteins Arf6: ADP-ribosylation factor 6

SNX4: sorting nexin 4

MICALL1: MICAL-like protein1 Ub : ubiquitinːqPcˑ

Eps15: epidermal growth factor receptor substrate 15 ESCRT: endosomal sorting complex required for transport

EGFR: Epidermal Growth Factor ReceptorːǶťʨČĥóİ²ˑ UBD: ubiquitin-binding domains

EEA1: Early endosome antigen 1

Hrs: hepatocyte growth factor regulated tyrosine kinase substrate

v-SNARE: vesicular-soluble N-etylmaleimide sensitive fusion protein attachment protein receptor

Snc1: SNAP receptor SNC1 COPI: coat protein I

WASH: Wiskott–Aldrich syndrome protein and SCAR homolog complex CF: Cystic fibrosisːĊȺŞȮȨǱˑ

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CFTR: Cystic Fibrosis Transmembrane conductance Regulator ∆F508: delta F508

ERQC: ER quality controlːķȺ²ĄɸȘǢƱƭˑ ERAD: ER associated degradationːķȺ²ʬʐÑɠˑ

PMQC: plasma membrane quality controlːŎɸȿĄɸȘǢƱƭˑ

CFBE: human CF bronchial epithelial cellːCF ŠȷǧƞpgƻʖǶȢȺƦˑ RFFL: ring finger and FYVE like domain containing E3 ubiquitin protein ligase DN: dominant-negative

BioID: Proximity dependent biotin identification WT: wild type

GFP: green fluorescent protein PCC: Pearson Correlation Coefficient

Lamp1: lysosomal associated membrane protein 1 GM130: Golgi matrix protein 130 kD

CI-M6PR: cation-independent mannose 6-phosphate receptorːzl\-6-ʠó İ²ˑ

TEM: Transmission Electron MicroscopeːʌʕēʻĥˆŚʧˑ SEM: Scanning Electron MicroscopeːɹƤēʻĥˆŚʧˑ MCC: Mander’s correlation coefficient

TfR: transferrin receptorːg\sJóİ²ˑ NT: Non-transfected

GPI: glycosylphosphatidylinositol NA: NeutrAvidin

CID: chemical-induced protein dimerization assay MB: Myc-Biotin

HB: histidine-biotin

GST: glutathione-S-transferase

LC-MS/MS : liquid chromatography coupled to tandem mass spectrometry KO: knockout

KD: knockdown

MYO1B: myosin IB MYO6: myosin VI MYO1E: myosin IE

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KIF5B: kinesin family member 5B KIF16B: kinesin family member 16B

RNF34: ring finger protein 34 (known as CARP1: caspases‑8/10 associated RING proteins 1)

XIAP: X-linked inhibitor of apoptosis

SH3RF1: SH3 domain containing ring finger 1 (known as POSH: Plenty of SH3s) DTX3L: deltex E3 ubiquitin ligase 3L

DUB: Deubiquitinating enzymesːȼqPcäʟȠˑ

VCPIP1: valosin containing protein interacting protein 1 (kown as VCIP135) USP15: ubiquitin specific peptidase 15

USP43: ubiquitin specific peptidase 43 SQSTM1: sequestosome 1

FBS: Fetal bovine serum

DMEM: Dulbecco's modified Eagle's medium MEM: minimum essential medium

PEI: Polyethylenimine

PBS (-): Ca2+_{, Mg}2+_{free Phosphate Buffered Saline}

PBS: Phosphate Buffered Saline BSA: Bovine serum albumin BCA ǀ: bicinchoninic acid assay SDS: Sodium dodecyl sulfate

PAGE: polyacrylamide gel electrophoresis HRP: Horseradish peroxidase

EDTA: ethylenediaminetetraacetic acid gRNA: guide RNA

PCR: Polymerase Chain Reaction PMSF: phenylmethylsulfonyl fluoride Tris: tris(hydroxymethyl)aminomethane

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Ǹ

ǸƳ

ȕ 1 ȓ ȭɰ ... 1 ȕ 2 ȓ RRFFL Ğǭ²Dǥ%ƱȻɠơ ... 9 ȕ 1 ș ƚ ȓ / Ǹ ǵ ... 9 ȕ 2 ș RFFL ȢȺËĹĐɠơ ... 9 ȕ 3 ȓ RRFFL-DN Ğǭ²Kh`|ƱȻ.ï6Őˀ ... 11 ȕ 1 ș ƚ ȓ / Ǹ ǵ ... 11 ȕ 2 ș RFFL-DN Ğǭ²Kh`|.=6Őˀ ... 11 ȕ1 ˁ RFFL-DN Ğǭ²ȢȺËĹĐɠơ ... 11 ȕ2 ˁ RFFL-DN Ğǭ²Kh`|ŎŤ.=6Őˀ ... 16 ȕ 3 ș RFFL-DN Ğǭ² RE ƱȻ.=6Őˀ ... 18 ȕ1 ˁ RFFL-DN Ğǭ² EE > RE 3/Ñʹ.=6Őˀ ... 18 ȕ2 ˁ RFFL-DN Ğǭ²ȏɉʃʊ.ï6Őˀ ... 20 ȕ 4 ȓ R\aä ERC Ŏť/Ñĥ}Nj]|/ɠƑ ... 26 ȕ 1 ș ƚ ȓ / Ǹ ǵ ... 26 ȕ 2 ș BioID ǀDǥ% RFFL ȥøČĥȩȲǵɠơ ... 26 ȕ 3 ș RFFL ȥøČĥ.=@R\aä ERC ŎťƱƭ/ɠơ ... 29 ȕ 5 ȓ RRFFL .=@ Rab11-effector Ub ÖŘƱƭ/ƪɣ ... 38 ȕ 1 ș ƚ ȓ / Ǹ ǵ ... 38 ȕ 2 ș In cell .@ RFFL .=@ Rab11-effector / Ub ä/ƪɦ . 38 ȕ1 ˁ RFFL Rab11-effector / Ub ä.=6Őˀ ... 38 ȕ2 ˁ Rab11-effector / Ub ä.@ RFFL /ǤǢǵƱȻ ... 40 ȕ3 ˁ TfR XHRS.@ Rab11-effector / Ub ä/ǤǢǵƱȻ .. 40 ȕ 3 ș In vitro .@ RFFL .=@ Rab11-effector / Ub ä/ƪɦ43 ȕ 6 ȓ ȶĲ ... 46 ȕ 1 ș RFFL-DN Ğǭ².@R\aä ERC ŎťÑĥƱƭ ... 46 ȕ 2 ș RFFL-DN Ğǭ².@ȏɉXHRSÖŘ ... 47 ȕ 3 ș RFFL-DN Ğǭ².@ EE > RE 3/ÑʹÖŘ ... 48 ȕ 4 ș RFFL Dý9 E3-ligase .=@ Rab11-effecor / Ub äÖŘ ... 49

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6 ȕ ȕ 5 ș Rab11-effector Ub ä.=@XHRSÖŘ ... 49 ȕ 6 ș Rab11-effector Ub äÖŘƱƭ/£Ŕ/ǳĺ ... 50 ȕ 7 ȓ ȫű ... 51 ȕ 8 ȓ Ĭˋ/ʜ ... 53 ȕ 1 ș Ĭ ˋ ƛ Ɖ ... 53 ȕ 2 ș Ĭ ˋ Ƌ ǀ ... 57 ȕ1 ˁ ȢȺĕˉǀ ... 57 ȕ2 ˁ ʛ«ĥĶÈǀ ... 57 ȕ3 ˁ ÆǮɏÅƣɈǀ ... 58

ȕ4 ˁ Transferrin uptake assay ... 59

ȕ5 ˁ Time-lapse imaging of TfR recycling ... 59

ȕ6 ˁ CFTR uptake assay ... 60

ȕ7 ˁ CD59 uptake assay ... 60

ȕ8 ˁ Dextran uptake assay ... 60

ȕ9 ˁ EGF uptake assay ... 61

ȕ10 ˁ Pull-down assay ... 61

ȕ11 ˁ Western blotting ... 61

ȕ12 ˁ In cell Ubiquitin assay ... 62

ȕ13 ˁ CID assay ... 63

ȕ14 ˁ Cellular localization analysis of biotinylated proteins ... 63

ȕ15 ˁ BioID assay ... 63

ȕ16 ˁ Silver stain ... 64

ȕ17 ˁ Establishment of RFFL KO cells by CRISPR/CAS9 system ... 64

ȕ18 ˁ TfR recycling assay ... 65

ȕ19 ˁ Protein purification ... 65

ȕ20 ˁ In vitro ubiquitination assay ... 65

ȕ21 ˁ Statistical analysis ... 67

ȕ22 ˁ Transmission electron microscopy (TEM) ... 67

ȕ23 ˁ Scanning electron microscopy (SEM) ... 67

ȕ24 ˁ Liquid chromatography coupled to tandem mass spectrometry ... 67

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7 î

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1

ȕ

1ȓ ȭɰ

KhXHgZ\0ȢȺğǛɸ/ȢȺË3/ò?ʆ8<˒ZSióİ² -,/ȿaoRɸ/bIQZD¤)˒ȢȺźǻ˒ȢȺʓɹ˒ ȢȺƬŞ=2ZSi«ʗ-,˒ĠƮ-ȢȺu^\Dɯș˒ȢȺşŁŞ /ȨŲ.Ŝ˂/őÜDƢ%1, 2˓KhXHgZ\.0˒R\¸Ħǵ

KhXHgZ\ːclathrin-mediated endocytosis: CMEˑ+R\ʽ¸Ħǵ KhXHgZ\ːclathrin-independent endocytosis: CIEˑ/˖(/ǭ-@Ʈŉ ĦĐ@˒KhXHgZ\A%ķȺ0˒#/Ʈŉ.C> ˒7 ÓƗKh`|ːearly endosome: EEˑ3+ʔ1A@3ːFig. 1-ˑ˓#/Ŕ˒ ķȺË/ȏɉ0˒EE >ŔƗKh`|ːlate endosomes: LEˑDȤ)`` |ːlysosomes: LYˑ3+ʃʊA@+``|ÑɠDó@ː``| ÑɠȤɼˑːFig. 1-ˑ3˓Ƌ˒EE >g\W[kdgRːtrans-Golgi network: TGNˑ<XHRSKh`|ːrecycling endosome: REˑ3+ʃ ʊA@+˒Ì2Ŏɸȿːplasma membrane: PMˑ3+Ũ?XHRSA

@ːXHRSȤɼˑːFig. 1- ˑ4,5_{˓XHRSȤɼ/ÕǗ0˒}_PM

>KhXHgZ\%ȏɉDÑɠ@+-Ì2 PM 3+ʃʊ@+

*˒ȏɉ/ÌøťD-*Ǐ9Ǘ.@˓EE > PM 3+ȏɉDXHR @Ȥɼ0˖(?˒EE >ǹź PM 3+Ũ@Ȥɼːfast recycling pathwaysˑːFig. 1-ˑ+˒ƧʇÀ.ĹĐä@ ERCːendocytic recycling compartmentˑDȤǧ )PM 3+Ũ@Ȥɼːslow recycling pathwaysˑːFig. 1-ˑǼ>A)@6˓

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Figure 1. The endosome/lysosome pathway

Endocytosed cargo is usually delivered to the EE that receives incoming material from primary vesicles generated by CME and CIE.

Cargoes are delivered from the EE to the LE and LY for lysosomal degradation. Cargoes are delivered from the EE to the TGN or to RE that brings the cargo back to

the PM.

Sorting of membrane proteins from EE leads to the entry of cargoes into fast recycling pathways.

Cargoes are trafficked via slow recycling pathway, which involves traffic through a juxtanuclear endocytic recycling compartment (ERC) and then via the RE before return to the PM. Endocytic Vesicle Lysosome (LY) Early Endosome (EE) Recycling Endosome (RE) Endocytic Recycling Compartment (ERC) Late Endosome (LE) CIE CME clathrin Golgi Trans-Golgi network (TGN) Cis Golgi Fast Slow Slow Cargo Early E Endosome E (EE) Late Endosome (LE) Lysosomal and Degradation Pathways E Fast Recycling Pathways Plasma Membrane (PM)

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3

Slow recycling pathway .)˒ERC 0 EE ťǚä.¬')ctǝ/ȿ

ʨŎťA˒#/ƶ?LE =2ĠȺ²ːmultivesicular bodies: MVBsˑ3

+ȋɒ)ːFig. 2ˑ2˓EE > LE 3/Kh`|ťǚä0 small GTPase *@Rab5 > Rab7 3/ĞäːRab5-Rab7 switchˑ+Kh`|/ʠŞä.

=?ɺ?˒/Kh`|ťǚä+Ê.ȏɉaoRɸ0EE > LY 3ʃ

ʊA@ːFig. 2ˑ3˓Kh`|ťǚä.=?˒EE /ȿ.ĹĐä@ȿa

oRɸEE Ë3+ËȽäA˒ȽËȿķȺːintraluminal vesicles: ILVsˑŎ ťA˒MVBs ǤťA@ːFig. 2ˑ3_{˓Small GTPase *@ Rab11 0 ERC /}

zN*?7˒Rab5 ʲŞ/ EE > Rab11 ʲŞ/ ERC 3/ȋɒːťǚäˑ

0əʸ*˒ATPase sE{aoRɸ*@ eps15 homology domain containing proteinsːEHDsˑ˒Rab11 KsJRaČĥ*@ Rab11-family interacting proteins ːRab11-FIPsˑ˒°Ñĥʤ G aoRɸ*@ ADP-ribosylation factor 6ːArf6ˑ˒ Sorting Nexin sE{aoRɸ*@ Sorting Nexin 4ːSNX4ˑ˒~aa

oRɸ*@Dynein˒Rab sE{°ÑĥʤaoRɸ Rab10˒Rab22A .

=')ÖŘA)@ːFig. 2ˑ2,8˓7%˒ERC > PM 3/ʃʊ0˒Arf6 ¸Ħ

ǵ.ŎťA%ctǝ/RE 2<EHD1 ¸Ħǵ.ŎťA%ctǝ/ RE

8_{-,əƇ/ȤɼĦĐ@ːFig. 2ˑ˓>.˒slow recycling pathway .@}

RE /ʃʊ0˒RE .ĹĐä@ Rab11 +~aaoRɸD Rab11-FIPs Ĥ ¤@+*ÖŘ)@ːFig. 2ˑ9_{. MICAL-like protein1ːMICALL1ˑ¸Ħǵ}

.RE .ĹĐä% EHD1 ;7% Rab11-FIP2 +Ǻ´ǥ@+>˒ RE Ʊ

Ȼ0 MICALL1˒EHDs˒Rab11-FIPs /éɯǵ-Á.=')ÖŘA)@+

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4

Figure 2. Molecular mechanisms of the endosome/lysosome pathway

LY LE/MVBs EE ERC PM Rab11

pH

6.8 4.5 ILVs Rab5 Rab7 Endocytic Vesicle Rab11 Rab11-FIP2 MYO5B EHD1 MICALL1 Arf6 Rab11 Actin EHDs Rab11-FIPs Arf6 SNX4 Dynein Rab10 Rab22A

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5

qPcːUbiquitin: Ubˑ0ȏɉ/KhXHgZ\˒Kh`|ʚÔ

ƱƭDÖŘ@ʢɛ-Čĥ*@ 10-12˓ȏɉ/ Ub ä0 Epsin =2 Epidermal

growth factor receptor substrate 15ːEps15ˑ.=@KhXHgZ\ 10 < endosomal sorting complex required for transportːESCRTˑəø².=@ MVBs 3

/ËȽäDɬĶ@ːFig. 3-ˑ13-15_{˓>.˒Kh`|ʃʊ;7%Kh}

`|ʬʐČĥ/ Ub ä.=?ÖŘA)@˓·1˒Eps15 / Mono-Ub ä

0Epidermal Growth Factor ReceptorːEGFRˑ/ȢȺËȋɒ<``|ʃʊD ÖŘ)?16,17˒Eps15 .ȥø% Mono-Ub +ɂɾ/ UBDːubiquitin-binding domainsˑȥø@+* Auto-inhibition ē+-? Eps15-UBD + Ub äDó

%ȏɉ+/ȥøʮĮDŊɺ-, 18Kh`|ťǚä.Ŝ˂*@ 17˓

7%˒EE /ɐø<Kh`|ťǚä.)eYSČĥ+)ƱȻ@

Early endosome antigen 1ːEEA1ˑ/ Mono-Ub ä;#/ƱȻ.Ŝ˂*@19˓ >.˒ESCRT aoRɸ*@ Hrs (Hepatocyte growth factor regulated tyrosine kinase substrate) MVBs 3ȏɉDËȽä@ʴ˒#/ƱȻ0 Ub ä.=')ɬ

ĶA@18 20˓A7*Ub 0 EE > LY 3/Kh`|ťǚäƱƭ.ʬ

@ + Ƈ Ġ ė ÿ A ) % ˓ ʇ ń ˒ ʟ Ƹ / v-SNARE (vesicular-soluble N-etylmaleimide sensitive fusion protein attachment protein receptor)

*@Snc1 (SNAP receptor SNC1) / Ub ä˒W[²Āʅ.ĦĐ@ķȺ/

ɔɜaoRɸCOPI (coat protein I) +/ȥøDÖŘ@+'%ėÿ<21_˒EE

>W[²3/ʋɒŞʃʊȤɼ/˕(*@gz¸ĦǵʃʊȤɼ.

)˒Kh`|.ĹĐä@ F-actin /ʢøƧŎťDÖŘ@ WASH

(Wiskott–Aldrich syndrome protein and SCAR homolog complex) /ƭʏĞä K63

ēUb ʦ.=')ÖŘA)@+'%ėÿ-AːFig. 3-ˑ22,23_{˒Ub /}

ȏɉXHRSZSi+)/ƱȻȃA%˓˒Rab11-effector

.=@ ERC D¤%XHRSƱȻÖŘƱƭ.@˒Ub /ʬ0É

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6

Figure 3. Role of Ub in endosomal sorting

Ubiquitination of cargo stimulates the lysosomal degradation by ESCRT complex. Reversible poly-ubiquitination of WASH, an actin-nucleating protein essential for

recycling, promotes endosomal protein recycling.

Figure 4. Degradation mechanism of ∆F508-CFTR

EGFR Ub EPS15 Hrs ESCR T ILV actin Auto-inhibition Active MAGE-L2 TRIM27 Arp2/3 Ubiquitination retromer WASH complex Ub Ub

EE

RE

PM

ER

unfolding

∆F Ubiquitination

degradation

CFTR

∆F508

CFTR

corrector

CF

∆F ∆F Ubiquitination

Reveal to

PMQC

mechanism

1

2

3

4

5 CFTR

stabilizer

target

6

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7 ĊȺŞȮȨǱːCF˘Cystic fibrosisˑ0ǴɈ¡ȍʫ*Ɣ;˄ņˍŁƣɈ² àŞʛ«ŞǯŠ*@24,25˓Ǳǝ+)ĂþĉţƣǱǉäĉʩę.Ŵ> A˒#/ƿǲǀ+)0ŬǤǛɸƻȘƂŰŌɍ-,/ĳǱǲǀ0@˒ˍ ūʤŜɛ*?˒ȬɀɊ-,/ĠÚȸŞć˅+-')@%:˒ƨƚƿ ǲɍ/ʪǳƖ7A)@26,27˓ƚǯŠ/íČ0˒ȢȺ/Ŏɸȿ*ĚȠHMc

k+)ÁCFTRːCystic Fibrosis Transmembrane conductance Regulatorˑ

/ƱȻǭŁ*@+Ǽ>A)@28˓ʍŁ˒CFTR 0ķȺ²*sLfG

SAŎɸȿ3+ʔ1A˒#/ƱȻDǳż@29˓˒CF Šȷ.)

0CFTR ʛ«ĥĞǭː90%§0∆F508 Ğǭˑ.=@ƭʏǭŁ>ķȺ²Ąɸ

ȘǢƱƭːERQC: ER quality controlˑ.=?˒ÑɠZSi*@ Ub äDó, ueF`|D¤%ķȺ²ʬʐÑɠːERAD : ER associated degradationˑ.

ʊ>A@ːFig. 4-ˑ30. #/%: CFTR ȢȺŎɸȿ.ǳǡ! ǳǰ.Ƀ@˓

7%˒ʇńCF /ƿǲɍ+)ʪǳA)@ CFTR VRa0˒ʜÑǵ.

∆F508-CFTR Ğǭ²/ȢȺȿ/ǳǡʤDƃĈ@ːFig. 4-ˑ31_˒VRa

.=@Ŏɸȿ.ʊ>A%∆F508-CFTR 0ŎɸȿĄɸȘǢƱƭːPMQC: plasma membrane quality controlˑ.=? Ub äDó˒ʎ<.``|ÑɠA@

%:ːFig. 4- ˑ˒ƿǲâƢ0ŋ˒çÑ-ɁŅâƢ0ŗ>A)-32_˓7%˒ ∆F508-CFTR Ğǭ²/ȢȺȿ>/ÑɠƱƭ0Ƙ&Ƒ-ʜÑĠ˒ƕâ-CF ɍǛǲǀʪǳ/ġ-ʵĜ+-')@ːFig. 4-ˑ33_{˓#*ÄɒǾȐ.} )˒ĊȺŞȮȨǱ/Ɗɞƿǲǀ/ʪǳDȐƬ/Ǹǵ+˒∆F508-CFTR Ğǭ² /PMQC .=@ÑɠƱƭ/ɠƑːFig. 4-ˑ=2ƊɞƿǲɍaUdg/Ź ȡɒCA%ːFig. 4-ˑ34_{˓Ŏɸȿ*/∆F508-CFTR Ğǭ²/ÑɠDʮĮ} @ƊɍDʪǳ*A1˒CFTR VRa+/ɍǛµǥǲǀ.=@ƿǲôȻ* @34. #/%:, 7 Ó:.˒PMQC .@ Ub äƱƭ/ÉɴɠƑDǸų˒

CF ŠȷǧƞpgƻʖǶȢȺƦːCFBE: human CF bronchial epithelial cell lineˑ

Dǥ%siRNA ȩȲǵ\RjSːE3 ligase: 636 ȍˇˑɒCA%34_˓#

/ȥƢ˒∆F508-CFTR Ğǭ²/ŎɸȿǳǡDŪÖ@Kh`|ĹĐqPc

O_RFFL (ring finger and FYVE like domain containing E3 ubiquitin protein

ligase) ùīA%34˓#*˒RFFL /ƱȻɠƑɒCA˒RFFL 0Ŏɸȿ<

Kh`|.ĹĐ@∆F508-CFTR Ğǭ²DʚŮǵ. Ub ä@+Ƒ>

+-?˒ƍĦɍ+/Ǻ´ǥDƗœ*@PMQC DƯǵ+% CF ɍǛƿǲǀ

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8 #*˒ƚǾȐ*0Kh`|.@RFFL />-@ƱȻɠơDǸǵ+ ˒ȍ /ƪɣDɒ'%˓7 ˒ȕ2 ȓ*0 RFFL /ĹĐäh}Hï2 RING ːUb ǄŞˑh}H.ĞǭDĶÈĹĐɠơDɒ'%˓#/ȥƢ˒RFFL Ub ligase ǄŞäĞǭ²*@dominant-negativeːDNˑĞǭ²0˒Kh`|ŎŤǭ ŁDŊɺ%˓#*˒ȕ3 ȓ*0 RFFL DN Ğǭ²Kh`|ƱȻ.

=6Őˀ/ƪɣDɒ'%˓7%˒ȕ4 ȓ*0 Proximity dependent biotin identification ːBioIDˑǀDǥ% RFFL ȥøČĥȩȲǵɠơDɒ˒R\a

äERC Ŏť/Ñĥ}Nj]|ɠƑDɨ8%˓ƔŔ.˒ȕ 5 ȓ*0ùī%

RFFL ȥøČĥ/ RFFL .=@ Ub ÖŘƱƭ.()ƪɣDɒ'%˓ §˒÷ȓ*ŗ>A%ǼɝDɪʉ@˓

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9

ȕ

2ȓ RFFL Ğǭ²Dǥ%ƱȻɠơ

ȕ1ș ƚȓ/Ǹǵ RFFL 0 PM =2Kh`|.ĹĐ˒ƭʏǭŁȿaoRɸ*@ ∆F508-CFTR D Ub ä@+*˒``|ÑɠȤɼ3/ʃʊDºʑ@34˓ #*˒RFFL /ĹĐäh}Hï2 Ub ǄŞäh}H/Ğǭ²Dǥ˒RFFL />-@ƱȻɠơDɒ'%˓ ȕ2ș RFFL ȢȺËĹĐɠơ RFFL 0 N ƙȔ.o{gHäXHgːC5, C6, C10ˑD¤)Kh`

|.ĹĐ˒C ƙȔ/ RING h}H.=? Ub äDɒ')@ːFig. 5Aˑ34˓

#*˒GFP (green fluorescent protein) tag ɐø RFFL ÷ȍĞǭ²ǳǡu\{h DƭȚ˒HeLa ȢȺ.ysJRZǀ.=?ʕŞ.ǳǡ!˒#/ȢȺ

ËĹĐDÊǙǗYˆŚʧ.=?ɠơ%˓RFFL-WT (wild type) -GFP 0K

h`|.ĹĐä%/.ĳ)˒ĹĐä.Ŝ˂- N ƙȔDƲž%Ğǭ²

ː∆2-88, ∆2-10ˑ0ȢȺɸ.ĹĐä%ːFig. 5Bˑ˓Ƌ˒RING h}HƲž ː∆RINGˑ0ĹĐ.Őˀ-'%ːFig. 5Bˑ˓Ʌāǋ+.˒RING h}H

/ǄŞś.ǗĞǭDĶÈ%RFFL-DN Ğǭ²ːH333A, 2CAˑ0ĹĐ

ȗũ.ʷȏ@+C'%ːFig. 5Bˑ˓#*˒RFFL-DN Ğǭ²/ N ƙȔ ƲžĞǭ²ːC5610A-2CA, ∆2-88-2CA, ∆2-10-2CAˑ/ĹĐDɠơ@+ȗũ .ʷȏ@+-ȢȺɸ.ĹĐä%ːFig. 5Bˑ˓Ŗ')˒RFFL 0Kh`

|)Ub ǄŞDƲĢ@+˒Kh`|ŎŤǭŁDŊɺ+ȶ

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10

Figure 5. RFFL catalytic inactive mutants induce condensed endosomes.

(A) Predicted function of RFFL domains. (B) Fluorescence micrographs of HeLa F508-CFTR-3HA cells expressing RFFL-GFP variants.

RFFL-WT ∆NT (2-88) ∆RING (313-363)

H333A 2CA(C316A,C319A) ∆NT(2-88)-2CA ∆2-10-2CA C5610A-2CA

∆NT (2-10)

B

FYVE-like RING 363 aa

RFFL 44 92 312 356 5 MWATCC6NWF10C Palmitoylation site 319 316 333

RING-domain point mutation site

A

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11

ȕ

3ȓ RFFL-DN Ğǭ²Kh`|ƱȻ.ï6Őˀ

ɏÅaoRɸɐøMOkzND co-transfection ˒ÊǙǗY ˆŚʧDǥ)ÊĹĐɠơDɒ'%. PCC (Pearson Correlation Coefficient) ɠơ

ï2HyVolutionːLeicaˑ.=@ɻɠÂH}[SDǥ% Line scan ɠơ.=

?RFFL-WT 0 EE zN*@ Rab5ːPCC: 0.438ˑ< EEA1ːPCC: 0.304ˑ˒ LE zN*@ Rab7ːPCC: 0.467ˑ< Lamp1 (lysosomal associated membrane protein 1)ːPCC: 0.442ˑ+ʜÑǵ.ÊĹĐ@+C'%ːFig. 6A and Bˑ˓

7%˒RE zN*@ Rab11ːPCC: 0.813ˑ+0ġʜÑÊĹĐ%ːFig. 6Aˑ˓

Ƌ˒RFFL-DN Ğǭ²0 Rab5ːPCC: 0.568ˑ˒EEA1ːPCC: 0.758ˑ˒Rab7ːPCC: 0.661ˑ˒Lamp1ːPCC: 0.457ˑ˒Rab11ːPCC: 0.946ˑ˒trans-Golgi zN*@ TGN46ːPCC: 0.567ˑDƧʇÀ3+ʷȏ!%ːFig. 6A and Bˑ˓ER zN* @Sec61ːPCC: -0.228ˑ<˒LE > TGN 3ʃʊA@zl\-6-ʠó İ²ːCI-M6PR: cation-independent mannose 6-phosphate receptorˑːPCC: 0.156ˑ˒ cis-Golgi zN*@ GM130 (Golgi matrix protein 130 kD) ːPCC: -0.104ˑ. 0ŐˀD=6-'%ːFig. 6Bˑ˓HyVolutionːLeicaˑ.=@ɻɠÂH}

[SDǥ%Line scan ɠơ.=?ɪȢ.ɠơ%ȥƢ˒RFFL-DN Ğǭ²0˒

Rab5˒EEA1˒Rab7˒Lamp1˒TGN46 +0ʜÑǵ.ÊĹĐ)?˒Rab11 +0 56ĪÉ.ÊĹĐ%ːFig. 6A and Bˑ˓7%˒ÆǮɏÅƣɈǀDǥ)ËĐŞ

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12

/Lamp1ːPCC: -0.063ˑ+/ÊĹĐDɟĲ%+B˒RFFL-DN Ğǭ².=@

Kh`|ŎŤǭŁ/Ā?.R\aSä%ːFig. 6Bˑ˓§/+

>˒Kh`|ŎŤǭŁ0LY .Ď7A% RE *?˒RFFL / Ub ǄŞ RE

ƱȻDÖŘ)@ôȻŞȃĆA%˓

Figure 6. RFFL catalytic inactive mutants are localized to recycling endosomes. (-continued) mRFP-Rab5 mRFP-Rab7 HyVolution RFFL-GFP H333A-GFP Zoom merge RFFL-GFP H333A-GFP 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL Rab5 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL Rab7 μ 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A Rab7 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A Rab5 PCC: 0.438 PCC: 0.467 PCC: 0.568 PCC: 0.661

A

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13

Figure 6. RFFL catalytic inactive mutants are localized to recycling endosomes. (-continued) DsRed-Rab1 1 Lamp1-RFP _RFFL-GFP H333A-GFP RFFL-GFP H333A-GFP 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL Rab11 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL Lamp1 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A Rab11 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A Lamp1 PCC: 0.813 PCC: 0.442 PCC: 0.946 PCC: 0.457

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14

Figure 6. RFFL catalytic inactive mutants are localized to recycling endosomes. (-continued) 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A EEA1 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A Lamp1 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL EEA1 μ 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL Lamp1 α EEA1 RFFL-GFP H333A-GFP α Lamp1 RFFL-GFP H333A-GFP PCC: 0.304 PCC: 0.186 PCC: 0.758 PCC: −0.063

0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A TGN46 μ 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL TGN46 mCherry-TGN46 RFFL-GFP H333A-GFP PCC: 0.459 PCC: 0.567

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15

Figure 6. RFFL catalytic inactive mutants are localized to recycling endosomes.

(A) Cellular localization of RFFL-GFP and RFFL-H333A-GFP in HeLa-F508 CFTR-3HA cells was analyzed with the co-transfected organelle markers indicated. Circled regions were further deconvoluted (HyVolution). Boxed regions are enlarged (Zoom). Line scans show profiles of fluorescence intensity against line distance. Bars, 10

0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A Sec61 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL Sec61 RFFL-GFP H333A-GFP mCherry-Sec61 RFFL-GFP H333A-GFP α CI-M6PR RFFL-GFP H333A-GFP α GM130 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A CI-M6PR 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm H333A GM130 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL CI-M6PR 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm RFFL GM130 PCC: 0.144 PCC: 0.173 PCC: 0.283 PCC: −0.104 PCC: 0.156 PCC: −0.228

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16

and organelle markers was measured by Pearson’s correlation coefficient (PCC). (B) Cellular localization of RFFL-GFP and RFFL-H333A-GFP in HeLa F508-CFTR-3HA cells was analyzed with organelle markers indicated. Circled regions were further deconvoluted (HyVolution). Boxed regions are enlarged (Zoom). Line scans show profiles of fluorescence intensity against line distance. Bars, 10 nucleus was stained by DAPI. Colocalization of RFFL variants and organelle markers was measured by PCC.

ȕ2ˁ RFFL-DN Ğǭ²Kh`|ŎŤ.=6Őˀ

HyVolution ɠơ.=') RFFL-DN Ğǭ².=')ŎťA% RE ǭŁƭʏ²

ctǝ/MOk*@+ȃĆA%ːFig. 7Aˑ˓#*˒>-@ŚȢƭʏDɯ4@%:.˒ʌʕēʻĥˆŚʧːTEM: Transmission Electron MicroscopeˑDǥ RE ǭŁƭʏ²DɟĲ%˓RFFL-H333A DʕÛǳǡ%Ȣ Ⱥ.)˒ƧʇÀ.ctǝ/ķȺ/ʷȏɟĲA%ːFig. 7B-Eˑ˓7%˒

#/ʷȏ%ctǝķȺ/Ā?.0˒ʻĥıņ/ˍLY ĹĐä)?˒

Lamp1 /ÆǮɏÅƣɈǀ+ùƮ/ȥƢŗ>A%ːFig. 7B-E, Fig. 6Bˑ˓c tǝķȺ/ǹŒ070 nm *?˒XHRSKh`|/Ɇǵ-ǹŒ

ː5070 nmˑ+øɄ)%35˓Ŗ')˒RFFL-DN Ğǭ².=')ŎťA@

Kh`|ŎŤǭŁ0R\aä% ERC *@+ȶ@˓7%˒

HyVolution ɠơ+ùƮ.R\aä% ERC . 100300 nm / EE35_ǌĐ

@+;ɟĲA%ːFig. 7E, arrowˑ.

>.ɹƤēʻĥˆŚʧːSEM: Scanning Electron Microscopeˑ.=')R

\aä%ERC /ƳÃɻŚȢƭʏDɟĲ%˓TEM +ùƮ.˒ctǝ

ķȺ/R\aäƧʇÀ*ȀɫA˒#/Ā?.0 LY ;ĹĐä)%

ːFig. 7F-Hˑ˓SEM .=@ɠơ.=')˒/ƭʏ²0˒ʐȧ%ctǝ* ȩǝ/MOk*0-˒ctǝ/ķȺR\aä)@+ C'%ːFig. 7G and Hˑ˓

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17

Figure 7. Ultrastructure of RFFL-H333A induced condensed recycling endosomes.

(A) Super-resolution confocal micrograph (HyVolution) of the condensed tubular structures induced by RFFL-H333A-GFP in HeLa F508-CFTR-3HA cells. Circled regions were further deconvoluted (HyVolution). Boxed regions are enlarged (Zoom). Bars, 10 of HeLa F508-CFTR-3HA cells transfected with RFFL-H333A-GFP. Higher magnification views of the section are shown (C-E). Arrow and arrowhead show EE and the clustered ERC, respectively. N, nucleus. Bars, 10 m (B), 2 m (C), 1 m (D, E). (F-H) Scanning electron microscopy of HeLa F508-CFTR-3HA cells transfected with RFFL-H333A-GFP. Clusters of ERC (blue, arrowhead) are observed around the nucleus (red). Lysosomes (yellow) are located around the clustered ERC in the cytoplasm (purple). Higher magnification views of the section are shown (G-H). Bars, 1 m.

B C D

H F G

N

RFFL-H333A-GFP HyVolution Zoom

A

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18 ȕ3ș RFFL-DN Ğǭ² RE ƱȻ.=6Őˀ ȕ1ˁ RFFL-DN Ğǭ² EE > RE 3/Ñʹ.=6Őˀ RFFL-DN Ğǭ²Kh`|ŎŤ&*0-˒Kh`|ƱȻ.Őˀ Dï6ƪɣ%˓Kh`|/ƱȻ0 Rab aoRɸ.=?ɯșA) @ 36˓Kh`|ťǚä.)˒EE .ĹĐä@ Rab5 ǄŞäA

Kh`|ȿ>ɠʹ˒¦C?.Rab7 ǄŞäA LE ŎťA@37-39_˓

7%˒Kh`|.ǄŞä%Rab11 ĹĐä@+*˒RE < ERC Ŏť

A)3˓HyVolution Dǥ% MCC (Manders’ Colocalization Coefficients) ɠ ơ.)˒Mock =2 RFFL-WT ʕÛǳǡƒ˒Rab7 =2 Rab11 /ȟ 30%

Rab5 /ĹĐ@ EE >ɠʹ)@+C'%ːFig. 8A and Bˑ˓Ƌ˒

RFFL-DN Ğǭ²ʕÛǳǡƒ˒Rab7 0 RFFL-WT ʕÛǳǡƒ+ùƮ˒Rab5 /Ĺ

Đ@ EE >ɠʹ)%/.ĳ)˒Rab11 0ɠʹA Rab5 +56ÊĹ

Đ@+Ƒ>+-'%ːFig. 8A and Bˑ˓Ŗ')˒RFFL-DN Ğǭ²0 LE ťǚä.0ŐˀD=6 ˒EE > RE 3/ÑʹDʚŮǵ.ʮĮ@+ ȃĆA%˓

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19 mTagBFP2-Rab5 WT H333A HyVolution

merge Zoom (BFP & RFP)

mock WT H333A mock mRFP-Rab7

mTagBFP2-Rab5 mRFP-Rab11 merge HyVolution Zoom (BFP & RFP)

B 0 0.2 0.4 0.6 0.8 1 MCC (Rab7 or Rab1

1 overlapped with Rab5 )

mock WT H333A RFFL-GFP: _mock WT H333A

Rab5 & Rab7 Rab5 & Rab11

n.s.

n.s. n.s.

A RFFL-GFP

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20

Figure 8. RFFL-H333A inhibits segregation of EE and RE.

(A) Cellular localization of mTagBFP2-Rab5 and mRFP-Rab7 or DsRed-Rab11 in HeLa-F508 CFTR-3HA cells transfected with RFFL-GFP variants or empty vector (mock). Circled regions were further deconvoluted (HyVolution). Boxed regions are enlarged (Zoom). Bars, 10 correlation coefficient (MCC) values quantify the mRFP-Rab7 or DsRed-Rab11 overlapped with mTagBFP2-Rab5 in the transfected HeLa cells shown in A. Data represent means ± SE (n=20 cells per each condition). n.s., not significant, ∗∗p < 0.01.

ȕ2ˁ RFFL-DN Ğǭ²ȏɉʃʊ.ï6Őˀ

RFFL-DN Ğǭ²ȏɉ/ PM >/KhXHgZ\Ȥɼ.ï6ŐˀD

ƪɣ%˓7 ˒CME .=')KhXHgZ\A˒PM .XHR

SA@g\sJóİ²ːTfR: transferrin receptorˑ3/ŐˀDɯ4%˓

Alexa Flour 647-TfːA647-Tfˑ.) PM .ĹĐä@ TfR DƯɳ˒Time-lapse H}[S.=')KhXHgZ\Ŕ/ʃʊD~jaS%˓ Non-transfectedːNTˑ=2 RFFL-WT ʕÛǳǡȢȺ.)˒ȢȺɓʾ/ TfR

DA647-Tf .) 2.5 ƒʫƯɳ˒}fGI|cJ[Ŕ 1 ƒʫ chaseːT-0ˑ

@+TfR 0KhXHgZ\AƧʇÀ/ ERC .ĹĐä)%ːFig. 9Aˑ˓

Ƌ˒RFFL-DN Ğǭ²ʕÛǳǡȢȺ/ TfR 0R\aä% ERC .Ɍȏ %˓#/Ŕ˒Time-lapse H}[S.=')˕ƒʫ A647-Tf /ŴãD~ja S%ȥƢ˒NT =2 RFFL-WT ʕÛǳȢȺ/ A647-Tf ɏÅōņ0ŕ . ǐĸ)˒Ɣȣǵ. 46%7*ǐĸ%+>˒ƯɳAKhXHg Z\%TfR /èÑ PM .XHRSA%+ȃA%ːFig. 9Bˑ˓ Ƌ˒RFFL-DN Ğǭ²ʕÛǳǡȢȺ*0˒TfR 0 ERC .Ɍȏ)?˒ȢȺË /A647-Tf ɏÅ/ǉĢ0 NT =2 RFFL-WT ʕÛǳǡƒ+ƹʀ)ƕŢ.°˒ ƯɳAKhXHgZ\% TfR /XHRSDʒň!@+Dȃ

%ːFig. 9A and Bˑ˓7%˒TfR +ùƮ. RFFL-DN Ğǭ²ʕÛǳǡ0 CME ȏ

ɉ*@ WT-CFTR40_D _{ERC .Ɍȏ!˒XHRS/ʒňDŊɺ}

% ːFig. 9C ˑ˓ > . ˒ CIE . = ' ) K h X H g Z \ A @ ˒ GPI (glycosylphosphatidylinositol) FNēaoRɸ*@ CD59 ; RFFL-DN Ğ

ǭ²ʕÛǳǡ.=')ERC .Ɍȏ%41_{ːFig. 9Dˑ˓§/ȥƢ=?˒RFFL /}

(29)

ȷ/ȏɉ/XHRS.)ʢɛ-21 őÜDƢ%+ȃĆA%˓ Ƴ.˒``|ÑɠȤɼ3ʃʊA@ȏɉaoRɸ.ĳ@RFFL-DN Ğ ǭ²/ŐˀDɯ4%˓NT =2 RFFL-WT ʕÛǳǡƒ˒RFFL /Ėɸ*?ǹ źǺ´ǥUb ä@{\sLh PM aoRɸ r∆F508-CFTR34_0˒_{4 h} chase Ŕ``|Ñɠ.=')ǉĢ%˓˒RFFL-DN Ğǭ²ʕÛǳǡ. )r∆F508-CFTR /``|Ñɠ0ʒň%ːFig. 9Eˑ˓Ƌ*˒rlXH gZ\zN*@ Dextran / LY 3/ʃʊ.0˒RFFL-DN Ğǭ²ʕÛǳ

ǡ0Őˀ-'%ːFig. 9F and Gˑ˓ƔŔ.˒EGFR /KhXHgZ\Ȥɼ

.ï6ŐˀDɯ4%˓EGFR 0° EGF ǖņː1.5-10 ng/mlˑ*0 CME .='

)KhXHgZ\AXHRSȤɼ3+ʃʊA@˒ˍEGF ǖņ

ː100 ng/mlˑ*0 CIE .=?``|ÑɠȤɼ3+ʃʊA@42˓Ŭ EGFR

Ŭ²Dǥ%ÆǮɏÅƣɈǀ.=@ɠơ/ȥƢ˒° EGF ǖņː10 ng/mlˑ.=

?KhXHgZ\A%EGFR 0 RFFL-DN Ğǭ².=? ERC .Ɍȏ

XHRSʒň%ːFig. 9Hˑ˓˒ˍ EGF ǖņː100 ng/mlˑ.=?

KhXHgZ\A%EGFR 0 RFFL-DN Ğǭ².=@Ɍȏ0Ǥ LY 3

+ʃʊA%ːFig. 9Hˑ˓ˍ EGF ǖņː100 ng/mlˑ.=@ EGFR / LY ʃʊD >.ɯ4@%:.˒Alexa Fluor 568-EGFːA568-EGFˑ.=?Ưɳ% EGFR

+ EE =2 LY MOkzN+/ÊĹĐɠơDɒ'%˓NT =2

RFFL-WT ʕÛǳǡƒ/ȢȺ*0˒15 min chase Ŕ EEA1 +ÊĹĐ˒60 min Ŕ Lamp1 +ÊĹĐ LY 7*ʃʊA@+ȃA%ːFig. 9I-Lˑ˓7%˒RFFL-DN

Ğǭ²ʕÛǳǡ.);ùƮ.60 min Ŕ Lamp1 +ÊĹĐ LY 7*ʃʊA

@+ȃA%ːFig. 9I-Lˑ˓§/ȥƢ>˒RFFL-DN Ğǭ²0 RFFL .=

')ÖŘA)@∆F508-CFTR §ğ/``|ÑɠȤɼ.0Őˀ! ˒ERC >/XHRSȤɼDʒň!@+ȶ>A@˓

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22

Figure 9. RFFL-H333A inhibits cargo recycling from the ERC (-continued)..

A

A647-Tf T-0 h T-1 h NT RFFL-GFP A647-Tf RFFL -GFP H333A -GFP A647-Tf T-0 h T-1 h T-1 h T-0 h A647-Tf H333A-GFP A647-Tf 0 25 50 75 100 0 0.25 0.5 0.75 1 A647-Tf intensity (%) chase time (h) NT (n=31) RFFL-GFP (n=11) H333A-GFP (n=12)

B

C

αHA (CFTR) αHA (CFTR) H333A-GFP αHA (CFTR) RFFL-GFP 0 h (2.5 h load) 4 h chase RFFL- GFP H333A- GFP αHA (CFTR)

D

αFlag (CD59) H333A-GFP αFlag (CD59) 0 h (4°C label) 4 h chase αFlag (CD59) αFlag (CD59) RFFL-GFP RFFL- GFP H333A- GFP

E

H333A-GFP αHA (r∆F508) αHA (r∆F508) αHA (r∆F508) RFFL-GFP αHA (r∆F508) 0 h (2.5 h load) 4 h chase RFFL- GFP H333A- GFP

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23

Figure 9. RFFL-H333A inhibits cargo recycling from the ERC (-continued).

F

TRITC-Dextran 1 h loading & 3 h chase Dextran 333A-GFP αLamp1 Dextran H333A-GFP Dextran αamp1

TRITC-Dextran 1 h loading & 3 h chase

RFFL-GFP Dextran αLamp1 Dextran RFFL-GFP Dextran αamp1 0 0.2 0.4 0.6 0.8 1 H333A WT NT

PCC (Dextran & Lamp1)

RFFL-GFP (24) (16) (35)

G

RFFL- GFP H333A- GFP

H

H333A-GFP αEGFR merge

0 ng/mL 10 ng/mL 100 ng/mL 1 h 1 h 1 h 0 0.25 0.5 0.75 1 0 4 8 12 Intensity μm H333A EGFR 0 0.25 0.5 0.75 1 0 4 8 12 Intensity μm H333A EGFR 0 0.25 0.5 0.75 1 0 4 8 12 Intensity μm H333A EGFR PCC−0.023 PCC: 0.439 PCC: −0.119

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24

Figure 9. RFFL-H333A inhibits cargo recycling from the ERC (-continued).

A568-EGF RFFL-GFP

200 ng/ml A568-EGF for 15 min 200 ng/ml A568-EGF for 60 min

αLamp1 RFFL-GFP A568-EGF A568-EGF αamp1 A568-EGF RFFL-GFP αLamp1 RFFL-GFP A568-EGF A568-EGF αamp1 20 568 E min K L 0 0.2 0.4 0.6 15 min 60 min

PCC (EGF & Lamp1)

(19) (24) (50) (29) (56) (22) A568-EGF αLamp1 H333A-GFP H333A-GFP

A568-EGF A568-EGF αamp1

A568-EGF αLamp1 H333A-GFP H333A-GFP A568-EGF A568-EGF αLamp1

EGFGFGFGFFF p11 A-GFGFGFGFGFFPP P F NT WT H333A RFFL- GFP H333A- GFP J 0 0.2 0.4 0.6 15 min 60 min

PCC (EGF & EEA1)

(53)(25)(24) (59)(27)(22) I A568-EGF αEEA1 333A-GFP H333A-GFP A568-EGF A568-EGF αEEA1 A568-EGF αEEA1 H333A-GFP H333A-GFP A568-EGF A568-EGF αEEA1 -EGFEGFGG A11 A-GFPGFGG

FPPPPP F A568-EGF

RFFL-GFP RFFL-GFP A568-EGF αEEA1 A568-EGF αEEA1 A568-EGF RFFL-GFP RFFL-GFP A568-EGF αEEA1 A568-EGF αEEA1 L-GFPGF 8-EGFG A11 FP F NT WT H333A RFFL- GFP H333A- GFP

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25

Figure 9. RFFL-H333A inhibits cargo recycling from the ERC.

(A, B) Time-lapse images of internalized TfR labeled with Alexa Fluor-647 conjugated Tf (A647-Tf) in Hela-F508 CFTR-3HA cells non-transfected (NT) or transfected with GFP-fused RFFL variants. Cells were loaded with A647-Tf for 2.5 hours at 37°C (T-0 h) and chased 1 hour at 37°C (T-1 h) to monitor the TfR recycling. Images of same cells at T-0 h and T-1 h were shown in A. Broken lines indicate contour of cell and nucleus. The intracellular A647-Tf intensity was quantified at the indicated time points (B). (C, D) Indirect immunostaining of WT-CFTR-3HA (C) and CD59-Flag (D) in HeLa cells transfected with GFP fused RFFL variants. Internalized WT-CFTR-3HA was labeled with anti-HA antibody for 2.5 hours at 37°C and cell surface CD59-Flag was labeled with anti-Flag antibody for 1 hour at 4°C. Immediately after the labeling (0 h) or after 4 hours chase at 37°C (4 h), cells were fixed and immunostained with secondary antibody. Asterisks and arrowheads indicate the NT cells and the condensed ERC, respectively. (E) Indirect immunostaining of rescued ∆F508-CFTR-3HA (r∆F508) in HeLa-∆F508-CFTR-3HA cells transfected with RFFL-GFP or RFFL-H333A-GFP. Internalized r∆F508-CFTR-3HA was labeled with anti-HA antibodies for 2.5 h at 37°C (T-0 h) and chased for 4 hours. Asterisks and arrowheads indicate the NT cells and the condensed ERC, respectively. (F, G) Lysosomal delivery of TRITC-Dextran loaded at 37°C for 1 hour and chased for 3 hours in HeLa cells transfected with GFP-fused RFFL variants.. Lamp1 was used as a lysosome marker. Colocalization of TRITC-Dextran with Lamp1 was quantified by calculating PCC (G). (H) Indirect immunostaining of EGFR in HeLa cells after EGF treatment at the indicated concentration for 1 hour. Line scans show profiles of fluorescence intensity against line distance. Colocalization of RFFL variants and EGFR was measured by PCC. Asterisks and arrowheads indicate the NT cells and the condensed ERC, respectively. (I, K) EGFR endocytic trafficking in HeLa cells transfected with RFFL-GFP or RFFL-H333A-GFP was monitored by Alexa Fluor-568 conjugated EGF (A568-EGF) loading for 15 min (left) or 60 min (right) with EEA1 (I) or Lamp1 (K) immunostaining. Asterisks and arrowheads indicate the NT cells and the condensed ERC, respectively. (J, L) Colocalization of A568-EGF with EEA1 (J) or Lamp1 (L) in HeLa cells transfected with RFFL-GFP variants was quantified by PCC. The number of cells from at least two independent experiments is indicated in parentheses. Data represent means ± SE. Bars, 10

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26

ȕ

4ȓ R\aä ERC Ŏť/Ñĥ}Nj]|/ɠƑ

ȕ1ș ƚȓ/Ǹǵ RFFL-DN Ğǭ²0 RFFL /Ėɸ*@ r∆F508-CFTR +ōď.Ǻ´ǥ@34_˓ Ŗ')˒RFFL-DN Ğǭ²0ƵŁ- ERC Ŏť.ʢɛ-Ñĥ+ōď.Ǻ´ǥ˒ #/ǤǢƱȻDʮĮ@ȥƢ˒ǭŁ-ERC /R\aäDɬĶ@ôȻŞ ȶ>A%˓#*˒RFFL ȥøČĥȩȲǵɠơDɒ+*˒R\aä ERC Ŏť/Ñĥ}Nj]|DɠƑ@++%˓ ȕ2ș BioID ǀDǥ% RFFL ȥøČĥȩȲǵɠơ BioID ǀ0˒Ŗƞ/ÆǮƽʯǀ+0ǭ-?˒ȥøaoRɸ.qMcDÊƕ ȥø@+*˒ƒǵ-Ǻ´ǥ<Śŋ-Ǻ´ǥDˍţņ.ƪÐ*@43˓

C ƙȔ. BirA*=2HA KrguDɐø% RFFLːRFFL- BirA*-HAˑĩī

ˍǳǡ CFBE ȢȺ.)˒ĕđ. 50M /qMcDǍß@+˒

RFFL-BirA*_{-HA +ÊĹĐ@qMcäaoRɸɟĲA%}_{ːFig. 10Aˑ}_˓

7%˒RFFL-H333A-BirA*-HA ĩīˍǳǡ CFBE ȢȺ.)˒R\aä ERC

ŎťA˒#.qMcäaoRɸɌȏ)%+>˒RFFL ȥø

Čĥ/qMcäȀɫA%ːFig. 10Aˑ˓7%˒IJ\atdeGS ǀ.=? RFFL-BirA*-HA =2 RFFL-H333A-BirA*-HA .=@ȥøČĥ/qM

cäDȀɫ%ːFig.10Bˑ˓#*˒NeutrAvidin FO\Dǥ)˒qM

cäaoRɸDȝɘʥƣɈDɒ'%ːFig.10Cˑ˓êʹ%qMcäa

oRɸDɸʤÑơǀ.=')Ñơ%ȥƢ˒RFFL ǜǭǵ.ȥø@ȟ 100 ȍˇ

/Kh`|ʬʐaoRɸDùī%˓#/*˒ƊɞRFFL ȥøČĥ+

)class I Rab11-FIPs (Rab11-FIP1˒Rab11-FIP2˒Rab11-FIP5)˒MICALL1˒MICALL2

ːJRABˑ˒EHD1Dý9ȟ30 ȍˇ/ RE ʬʐaoRɸːFig.10Dˑ˒7 ȍˇ/~

aaoRɸːFig.10Eˑ˒6 ȍˇ/ E3-ligaseːFig.10Fˑ˒3 ȍˇ/ DUBːFig.10Fˑ

Dùī%˓RE ʬʐaoRɸ/*˒Ɗɞ RFFL ȥøČĥ+)¯.Ŵ

>A%MICALL2 §ğ/˒class I Rab11-FIPs˒MICALL1˒EHD10TfR XHR

S.ʬ)@+Ǽ>A)%44-51˓#*˒A>/Čĥ.ǻǸ˒

(35)

27

Figure 10. RFFL interactome analysis by BioID (-continued).

A

αHA (RFFL)

SA-A568

(Biotinylated protein) merge

αHA (RFFL)

SA-A568

(Biotinylated protein) merge

RFFL- _BirA*-HA NT RFFL-H333A- BirA*-HA Biotin (-) Biotin (+) 70 210 70 55 40 20 15 10 NA-HRP (Biotinylated protein) αHA (RFFL) NT WT H333A input RFFL-BirA*-HA: (kDa) 210 90 55 40 35 140 70 (kDa) NT WT H333A NA Pull-down Silver Staining

B

C

D

Gene ID NT WT H333A RFFL RAB11FIP1 RAB11FIP5 SCAMP1 MICALL1 MICALL2 EHD1 RAB11FIP2 RAB11B RAB8A STX8 PACSIN2 ARFGEF2 NDRG1 LDLRAP1 VPS33B RAB10 EHD4 INPP5F VIPAS39 ITGB1 RAB13 RAB14 STX7 VPS50 UNC13D EHD2 VAMP8 ARF6 VTI1B

Total spectrum count

(36)

28

Figure 10. RFFL interactome analysis by BioID.

(A) Fluorescence micrographs of CFBE cells stably expressing RFFL-BirA*_{-HA or}

RFFL-H333A-BirA*_{-HA with or without 50}

biotinylated proteins were stained with anti-HA antibody and streptavidin-Alexa Fluor 568 (SA-A568). The non-transfected CFBE cells (NT) were used as a negative control. Bars, 50 confirmed the proximal biotinylated proteins and expression of RFFL-BirA*_{-HA or}

RFFL-H333A-BirA*_{-HA. Biotinylated proteins were detected with NeutrAvidin (NA)-HRP. (C)}

SDS-PAGE analysis of the BioID pull-down using the CFBE cells stably expressing RFFL-BirA*_{-HA or}

RFFL-H333A-BirA*_{-HA after 50}

identified with high confidence in RFFL-BirA*_{BioID. Heat maps represent total spectral counts of}

individual proteins per condition. (E, F) Comparison of the 7 motor proteins (E), 6 Ub ligase and 3 DUB (F) identified with high confidence in RFFL-BirA*_{BioID. The non-transfected (NT) CFBE cells were}

used for as a negative control. Heat maps represent total spectral counts of individual proteins per condition.

E

Gene ID NT WT H333A RNF34 XIAP SH3RF1 TRIM25 DTX3L SH3RF2 VCPIP1 USP15 USP43

0 100 Ub ligase DUB

F

Gene ID NT WT H333A MYO1B MYO6 MYO1E KIF5B KIF16B KIF13B KLC1

(37)

29

ȕ3ș RFFL ȥøČĥ.=@R\aä ERC ŎťƱƭ/ɠơ

BioID ǀ/ȥƢ/Ȁɫ/%:˒7 RFFL ȥøČĥ+ RFFL /ÊĹĐɠơD˒

PCC ɠơ.=?ɒ'%˓RFFL-mCherry + GFP-MICALL1ːPCC: 0.445ˑ˒-EHD1 ːPCC: 0.592ˑ˒-Rab11-FIP1C/RCPːPCC: 0.363ˑ˒-Rab11-FIP2ːPCC: 0.278ˑ˒ -Rab11-FIP5ːPCC: 0.265ˑ0ÊĹĐ%ːFig.11Aˑ˓>.šʍ?˒MICALL1 ːPCC: 0.717ˑ˒EHD1ːPCC: 0.723ˑ˒Rab11-FIP1CːPCC: 0.861ˑ˒Rab11-FIP2ːPCC: 0.952ˑ˒Rab11-FIP5ːPCC: 0.955ˑ0 RFFL-DN Ğǭ².=?ŎťA%R\

aä ERC .Ɍȏ%ːFig.11Aˑ˓7%˒HyVolutionːLeicaˑ.=@ɻɠÂH

}[SDǥ%Line scan ɠơ.); Rab11-effector 0 RFFL-DN Ğǭ²

.=?ŎťA%R\aäERC .Ɍȏ%ːFig.11Aˑ˓Pull-down assay .

)˒RFFL-DN Ğǭ²0 RFFL-WT =?; Rab11-effector +ōď.ȥø@ +C'%ːFig.11B-Hˑ˓RFFL-WT + Rab11-effector +/ÊĹĐɠơ+Ʉ )˒EHD1 0¥/ Rab11-effector +ƹʀ) RFFL-WT +ōǺ´ǥ% ːFig.11D-H ˑ˓ Æ Ǯ ɏ Å ƣ Ɉ ǀ . = ' ) ˒ RFFL-DN Ğ ǭ ² . = @ Ë Đ Ş Rab11-effector 3/ŐˀDƪɦ%+B˒ŎťA%R\aä ERC . MICALL1˒EHD1˒Rab11-FIP1C˒Rab11-FIP5 Ɍȏ%ːFig.11Iˑ˓Rab11-FIP2

.)0˒ËĐŞÑĥDɫɳ@Ŭ²Õǥ*-'%%:˒ɠơ*-'%˓A>/ȥƢ>˒RFFL-DN Ğǭ² Rab11-effector DKh`|

.gduɠʹDʮĮ@+#A>/ǤǢǵƱȻ/ʮĮDŊɺ˒

R\aäERC ŎťA@+¨ɮDȒ)%˓

#*˒#/¨ɮDĬɦ@%:.˒Rab11-effector D RFFL-WT .ōÖǵ. ȥø!@chemical-induced protein dimerization (CID) assay52_Dǥ@+*ƪ

ɦ%˓Rab11-effector . FRB-GFP D RFFL-WT . FKBP-mCherry Dɐø CID assay Dɒ'%˓FRB =2 FKBP / 2 ʤ²äDɬĶ@ozHZǍßÙ0˒ RFFL-WT =2 Rab11-effector 0Kh`|.ĹĐä)%ːFig.11J and Kˑ˓

Ƌ˒ozHZDǍß@+RFFL-WT + Rab11-effector ÊĹĐR\

aäERC ŎťA%ːFig.11Jˑ˓RFFL-DN Ğǭ²/ɓǡē+Ʉ)˒CID

assay .=?ɬĶA%R\aä ERC .˒KhXHgZ\A% WT-CFTR Ɍȏ%ːFig.11Lˑ˓Ŗ')˒RFFL Kh`|*Rab11-effector

Dgdu@+˒R\aäERC Ŏť=2ȏɉ/XHRSʮ

(38)

30

Figure 11. Prolonged RFFL association with Rab11 effectors induces the clustered ERC (-continued). HyVolution Zoom merge RFFL-WT -mChe H333A-mChe GFP-MICALL1 RFFL-WT -mChe H333A-mChe GFP-EHD1 RFFL-WT -mChe H333A-mChe GFP-Rab1 1-FIP1C 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm EHD1 H333A 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm Rab11FIP1 H333A

A

0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm MICALL1 H333A 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm MICALL1 RFFL 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm EHD1 RFFL 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm Rab11FIP1 RFFL PCC: 0.445 PCC: 0.592 PCC: 0.717 PCC: 0.723 PCC: 0.363 PCC: 0.861

(39)

31

Figure 11. Prolonged RFFL association with Rab11 effectors induces the clustered ERC (-continued). RFFL-WT -mChe H333A-mChe GFP-Rab1 1-FIP2 RFFL-WT -mChe H333A-mChe GFP-Rab1 1-FIP5 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm Rab11FIP2 H333A 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm Rab11FIP2 RFFL 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm Rab11FIP5 RFFL 0 0.25 0.5 0.75 1 1.25 0 1 2 3 4 5 intensity μm Rab11FIP5 H333A PCC: 0.278 PCC: 0.952 PCC: 0.265 PCC: 0.955

(40)

32

Figure. 11. Prolonged RFFL association with Rab11 effectors induces the clustered ERC (-continued).

B

293MSR 55 40 40 αV5 (RFFL) αV5 (RFFL) 70 55 140 αMyc EHD1 MICALL1 PD: NA-Agarose Input RFFL-V5: H333A WT H333A WT H333A WT - MICALL1 MB: EHD1 90 (kDa) PCC: 0.955

C

FIP1, 5 FIP2 40ー 35ー 90ー 70ー 40ー 35ー αV5 (RFFL) αMyc αV5 (RFFL) RFFL-V5: H333A WT WT _H333A WT _H333A - MB: _-FIP1CRab11 (kDa) _293MSR H333A WT Rab11 -FIP2 Rab11 -FIP5

PD: NA-Agarose Input

D

E

90 70 90 70 αGFP (EHD1) 70 55 40 NA-HRP (RFFL) PD: NA-Agaros RFFL-HB: - _WT H333A GFP-EHD1 αGFP (EHD1) 293MSR (kDa) Input 140 - H333A WT GFP-MICALL1 210 140 70 55 40 αGFP (MICALL1) NA-HRP (RFFL) αGFP (MICALL1) 293MSR RFFL-HB: (kDa) PD: NA-Agaross Input

F

αGFP (FIP1) NA-HRP (RFFL) RFFL-HB: - _WT H333A GFP-Rab11FIP1C αGFP (FIP1C) 293MSR (kDa) 90 70 55 40 90 PD: NA-Agarose Input 70 RFFL-HB: - _WT H333A GFP-Rab11FIP2 αGFP (FIP2) 70 55 40 NA-HRP (RFFL) 70 293MSR αGFP (FIP2) PD: NA-Agarose Input (kDa)

G

70 55 40 RFFL-HB: - _WT H333A GFP-Rab11FIP5 αGFP (FIP5) NA-HRP (RFFL) 90 70 αGFP (FIP5) 90 70 PD: NA-Agarose Input (kDa) 293MSR

H

(41)

33

Figure. 11. Prolonged RFFL association with Rab11 effectors induces the clustered ERC (-continued).

I

αMICALL1 αEHD1 αRab11-FIP1 αRab11-FIP5 H333A-mChe merge merge merge merge H333A-mChe H333A-mChe H333A-mChe

(42)

34

Figure 11. Prolonged RFFL association with Rab11 effectors induces the clustered ERC (-continued).

EHD1-FRB-GFP & RFFL-FKBP-mChe

control +Rap EHD1 RFFL EHD1 RFFL MICALL1 RFFL control MICALL1 RFFL +Rap

GFP-FRB-MICALL1 & RFFL-FKBP-mChe

YFP RFFL

control

YFP RFFL

+Rap

YFP-FRB & RFFL-FKBP-mChe

J

CFP EHD1 control +Rap CFP EHD1 EHD1-FRB-GFP & CFP-FKBP CFP MICALL1 control +Rap CFP MICALL1 GFP-FRB-MICALL1 & CFP-FKBP

K

(43)

35

Figure 11. Prolonged RFFL association with Rab11 effectors induces the clustered ERC (-continued).

FIP1C-FRB-GFP & RFFL-FKBP-mChe

FIP1C RFFL

merge

control +Rap

FIP5-FRB-GFP & RFFL-FKBP-mChe

RFFL FIP5 RFFL

+Rap

FIP5

control

FIP2-FRB-GFP & RFFL-FKBP-mChe

RFFL FIP2 control +Rap RFFL FIP2 +Rap CFP FIP1 control +Rap CFP FIP1C FIP1C-FRB-GFP & CFP-FKBP CFP FIP5 control +Rap CFP FIP5 FIP5-FRB-GFP & CFP-FKBP CFP FIP2 control +Rap CFP FIP2 GFP-FRB-FIP2 & CFP-FKBP

(44)

36

Figure 11. Prolonged RFFL association with Rab11 effectors induces the clustered ERC.

(A) Cellular localization of the transfected GFP-fused Rab11 effectors in HeLa-F508 CFTR-3HA cells was analyzed with the co-transfected RFFL-mCherry or RFFL-H333A-mCherry. Circled regions were further deconvoluted (HyVolution). Boxed regions are enlarged (Zoom). Line scans show profiles of

EHD1-FRB-GFP & RFFL-FKBP-mCherry (+Rap)

WT-CFTR _merge

EHD1 RFFL

2.5 h load

merge

L

FIP1C-FRB-GFP & RFFL-FKBP-mCherry (+Rap) WT-CFTR

FIP1C RFFL merge

2.5 h load

FIP5-FRB-GFP & RFFL-FKBP-mCherry (+Rap) merge

FIP5 RFFL WT-CFTR

2.5 h load

GFP-FRB-FIP2 & RFFL-FKBP-mCherry (+Rap) merge

FIP2 RFFL WT-CFTR

2.5 h load

GFP-FRB-MICALL1 & RFFL-FKBP-mCherry (+Rap) merge

MICALL1 RFFL WT-CFTR

(45)

37

fluorescence intensity against line distance. Colocalization of RFFL variants and Rab11 effectors was measured by PCC. (B, C) Interaction of Myc-Biotin (MB) tagged Rab11 effectors with RFFL-V5 or RFFL-H333A-V5 is shown by NA pull-down in 293MSR cells. (D-H) Interaction of histidine-biotin (HB) tagged RFFL or RFFL-H333A with GFP-Rab11 effectors is shown by NA pull-down in 293MSR cells. (I) Cellular localization of endogenous Rab11 effectors was analyzed by immunostaining with the indicated antibodies in HeLa- F508 CFTR-3HA cells transfected with RFFL-H333A-GFP. (J) Fluorescence imaging of the clustered ERC formation after forced RFFL-FKBP-mCherry association with Rab11 effectors-FRB-GFP in HeLa-F508 CFTR-3HA cells with or without 500 nM rapamycin (+Rap) treatment for 5 min followed by 16 hours chase after rapamycin washout. Broken lines indicate contour of cell and nucleus. (K) Negative control experiments of CID technique in HeLa-F508 CFTR-3HA cells transfected with Rab11 effectors-FRB-GFP and CFP-FKBP with or without 500 nM rapamycin (+Rap) treatment for 5 min followed by 16 hours chase after rapamycin washout. (L) Internalized WT-CFTR was accumulated in the clustered ERC induced by the CID in HeLa-CFTR-3HA cells expressing EHD1-FRB-GFP, GFP-FRB-MICALL1, Rab11-FIP1C-FRB-GFP, GFP-FRB-Rab11-FIP2 or Rab11-FIP5-FRB-GFP and RFFL-FKBP-mCherry with rapamycin treatment. Internalized WT-CFTR was labeled with anti-HA antibody as Fig. 9C. Bars, 10

(46)

38

ȕ

5ȓ RFFL .=@ Rab11-effector Ub ÖŘƱƭ/ƪɣ

ȕ1ș ƚȓ/Ǹǵ

RFFL-DN Ğǭ² Rab11-effctor Dgdu˒R\aä ERC ŎťDŊ

@Rab11-effector / Ub äÖŘDƪɦ%˓

ȕ2ș In cell .@ RFFL .=@ Rab11-effector / Ub ä/ƪɦ

ȕ1ˁ RFFL Rab11-effector / Ub ä.=6Őˀ

ȺDôǓäŔ˒NeutrAvidin .) Pull-down Dɒ˒IJ\atdeGS

ǀ.=?Ub ä/ƪÐDɨ8%˓#/ȥƢ˒Rab11-FIP5 §ğ/ MICALL1˒EHD1˒

Rab11-FIP1C˒Rab11-FIP2 .) Ub äƪÐA%ːFig. 12Aˑ˓>.˒#

.RFFL-DN Ğǭ²DʕÛǳǡ@+ Rab11-effector / Ub ä0Ýǵ.ʮĮ

A%ːFig. 12A-B and 11C-F, lane 4ˑ˓7%˒Rab11-effector / Ub ä0Ñĥʤ> mono-Ub ä*@ôȻŞȶ>A%˓#*˒Ub-K0 Ğǭ²ːÉ)/[

DFQj.ȱŻ mono-Ub ʦ0Ŏť*@;//˒poly-Ub ʦ0Ŏť*

-Ğǭ²ˑDǥ)ĬˋDɒ'%+B˒Rab11-effector / Ub ä0 WT-Ub D

¶ǥ%ʴ+ùƮ/Ñĥʤ.ƪÐA%+>˒A>/ġʜÑ0 mono-Ub

ä*@ôȻŞȶ>A%ːFig. 12C-F, lane 6ˑ˓7%˒liquid chromatography coupled to tandem mass spectrometryːLC-MS/MSˑ.=? MICALL1˒EHD1˒ Rab11-FIP1C˒Rab11-FIP2 / Ub äʜ¯Dùī%ːFig. 12Gˑ˓RFFL-WT Dʕ Ûǳǡ@+˒MICALL1 + EHD1 / Ub ä0Ğäǘ'%/.ĳ)˒ Rab11-FIP1C 0 poly- and/or multiple-Ub äěß%ːFig. 12C-F, lane 3ˑ˓Ʌā

ǋ+.˒Rab11-FIP2 .)0 poly- and/or multiple-Ub äÝǵ.ěß%

(47)

39

Figure 12. RFFL-H333A inhibits ubiquitination of Rab11 effectors.

(A, B) Ubiquitination level of MB-EHD1, MB-MICALL1, MB-Rab11-FIP1C, MB-Rab11-FIP2 and MB-Rab11-FIP5 in 293MSR cells transfected with HA-Ub and RFFL-H333A-V5 was measured by NA pull-down (A). Ubiquitination level of Rab11 effector proteins were quantified. At least 4 independent experiments were performed for quantification. Sample number was indicated in parentheses. Data represent means ± SE. *_P<0.05,***_{P<0.001 (B). (C-F) Ubiquitination level of MB-MICALL1 (C),}

MB-EHD1 (D), MB-Rab11-FIP1C (E) or MB-Rab11-FIP2 (F) in 293MSR cells transfected with HA-Ub or HA-Ub-K0 and RFFL-V5 or RFFL-H333A-V5 was measured by NA pull-down. (G) Ubiquitination sites of MB-MICALL1, MB-EHD1, MB-Rab11-FIP1C, and MB-Rab11-FIP2 in 293MSR cells transfected with HA-Ub were analyzed by LC-MS/MS. Three, five, ten and five ubiquitination sites were identified in the MICALL1, EHD1, Rab11-FIP1C, and Rab11-FIP2 respectively.

HA-Ub RFFL-H333A-V5 MICALL1 EHD1 Rab1 1-FIP1C Rab1 1-FIP2 Rab1 1-FIP5 MB- 210ー 140ー 90ー αMyc 70ー 140ー 90ー 70ー αHA (Ub) 210ーーーーー PD: NA-Agarose 55ー αV5 40ー 35ー Input (kDa) 293MSR A C 140 210 140 40 αV5 (RFFL) αMyc (MICALL1) Input PD: NA-Agarose αHA (Ub) (kDa) 1 2 3 4 5 6 HA-Ub HA-Ub-K0 MB-MICALL1 RFFL-V5 H333A-V5 D 70 90 140 70 40 αV5 (RFFL) Input

PD: NA-Agarose (EHD1) αMyc αHA (Ub) (kDa) 293MSR 1 2 3 4 5 6 HA-Ub HA-Ub-K0 MB-EHD1 RFFL-V5 H333A-V5 E Input PD: NA-Agarose (kDa) 293MSR αHA (Ub) αMyc (FIP1C) αV5 (RFFL) 1 2 3 4 5 6 HA-Ub HA-Ub-K0 MB-FIP1C RFFL-V5 H333A-V5 140ー 90ー 210ー 90ー 40ー 140ー 90ー 210ー 70ー 70ー 40ー F Input PD: NA-Agarose (kDa) 293MSR αMyc (FIP2) αV5 (RFFL) αHA (Ub) 1 2 3 4 5 6 HA-Ub HA-Ub-K0 MB-FIP2 RFFL-V5 H333A-V5 0 25 50 75 100 Ub level (α HA/ α Myc, % of Mock) MICALL1EHD1 Rab11 -FIP1CRab11 -FIP2

Mock H333A (9) (9) (9) (9) (12) (12) (5) (5) B Rab11-FIP2 (coverage 79.9%)

Site Identified peptide sequence K157 or K159 NNMTASMFDLSMKDK_{NNMTASMFDLSMK}GG_DKTRGGTR K374 RSDKGG_{LNNGGSDSPCDLK} K374 or K387 RSDK_{RSDKLNNGGSDSPCDLK}GGLNNGGSDSPCDLK GG

MICALL1 (coverage 72.7%)

Site Identified peptide sequence K250 QQHQQQLAEDAKGG K747 ESELIYVFKGG_QQNLEQR K782 EKGG_{VLMQELVTLIEQR}

EHD1 (coverage 94.5%)

Site Identified peptide sequence K32 QLYAQKGG_LLPLEEHYR K58 FHEFHSPALEDADFDNKGG_{PMVLLVGQYSTGK} K220 VVLNKGG_ADQIETQQLMR K280 KLFEAEEQDLFKGG K305 LAKGGVHAYIISSLKK LAKGG_VHAYIISSLK Rab11-FIP1C(coverage 83.8%)

Site Identified peptide sequence K33 AK_AKGGGGGPGGTSDAYAVIQVGKEK _{GPGGTSDAYAVIQVGK} K129 and K131 LKGG_SKGG_PGK K178 IKGG_GKNK K295 QLNQVNFTLPKGG K367 HLFSSTENLAAGSWKGG_EPAEGGGLSSDR K443 or K444 SSLLSLMTGKK_SSLLSLMTGKGGGG_KDVAKDVAK K578 KGG_{YSPSDPAFAYAQLTHDELIQLVLK} K644 IPTQVGKGG G

(48)

40

ȕ2ˁ Rab11-effector / Ub ä.@ RFFL /ǤǢǵƱȻ

RFFL /ǤǢǵƱȻDƪɦ@%:.˒CRISPR / CAS9 Z\e|.=') RFFL

KOːknockoutˑ 293MSR ȢȺDưȒːFig. 13A-Cˑ˒RFFL KO .@ Rab11-effector / Ub ä.ï6ŐˀDƪɦ%˓RFFL KO .=') Rab11-FIP1C

/Ub ä0ƕŢ.ǐĸ˒#. RFFL-WT DÌĶÈ@+* Ub ä/ǐĸ0

ċř%ːFig. 13D lane 6 and 7, and Fig. 13Eˑ˓Ƌ˒RFFL KO .=')˒MICALL1˒

EHD1˒Rab11-FIP2 / Ub ä0ǐĸ-'%ːFig. 13E, 12F-H lane 6ˑ˓Ʌāǋ +.˒RFFL KO ȢȺ. RFFL-DN Ğǭ²DʕÛǳǡ@+˒RFFL WT ȢȺ

+ùƮ.Rab11-effector / Ub äDǐĸ!%ːFig. 13D, 12F-H lane 8ˑ˓§/

ȥƢ=?˒ Rab11-FIP1C / Ub ä0. RFFL .=')ÖŘA)@ôȻŞ

ȶ>A@˓7%˒¥/Rab11-effector / Ub ä0 RFFL D KO );ǐĸ

-+>˒RFFL Dɗ=.¥/ E3-ligase ÖŘ)@ôȻŞȶ

>A@˓¥/E3-ligase .ʬ)0˒BioID ǀ.=? RFFL .ȥø@ E3-ligase

¾ɗ+)Ŵ>A@*BːFig.10Fˑ˓7%˒RFFL KO ȢȺ. RFFL-DN Ğǭ²DʕÛǳǡ%ʴ˒Rab11-effecor / Ub äǐĸ%+>˒RFFL-DN Ğǭ²Rab11-effector +ōď.ʨƒʫȥø@+*˒¥/ E3-ligase /ȥøD ʮĮ)@ôȻŞȃĆA%˓ ȕ3ˁ TfR XHRS.@ Rab11-effector / Ub ä/ǤǢǵƱȻ Ƴ.RFFL KO .@ TfR /XHRSDƪɦ%+B˒RFFL-DN Ğǭ²ʕÛǳǡƒ+0ǭ-?˒RFFL KO .=@ TfR XHRS/ʮĮ0Ȁ ɫA-'%ːFig. 13I, Jˑ˓Ƌ˒RFFL KO ȢȺ. RFFL-DN Ğǭ²DʕÛǳ ǡ@+˒ RFFL WT ȢȺ+ùƮ. TfR /R\aä ERC .@ɌȏȀ ɫA%ːFig. 13Jˑ˓§/+>˒ȏɉ/XHRS.)˒ Rab11-effector / Ub ä0Ŝ˂*@˒Rab11-FIP1C / Ub ä/8DʮĮ); çÑ*@++Ƒ>+-'%˓

(49)

41

Figure 13. RFFL regulates ubiquitination of the Rab11 effectors in cell (-continued). 1 2 3 4 5 6 7 8 HA-Ub MB-EHD1 RFFL-V5 H333A-V5 1 2 3 4 5 6 7 8 HA-Ub MB-MICALL1 RFFL-V5 H333A-V5 WT RFFL KO 90 140 70 90 70 40 αV5 (RFFL) αHA (Ub) αMyc (EHD1) Input (kDa) _293MSR PD: NA-Agarose 1 2 3 4 5 6 7 8 HA-Ub MB-FIP1C RFFL-V5 H333A-V5 WT RFFL KO Input 293MSR PD: NA-Agarose WT RFFL KO 140 210 140 210 90 40 αV5 (RFFL) αHA (Ub) αMyc (MICALL1) Input (kDa) 293MSR PD: NA-Agarose αV5 (RFFL) αMyc (FIP1C) αHA (Ub) (kDa) 1 2 3 4 5 6 7 8 HA-Ub MB-FIP2 RFFL-V5 H333A-V5 WT RFFL KO Input (kDa) PD: NA-Agarose αMyc (FIP2) αV5 (RFFL) αHA (Ub) 140ー 90ー 210ー 70ー 70ー 40ー 40ー 90ー 140ー 90ー 210ー F G D H 0 20 40 60 80 100 120 140 160 WT RFFL KO

MICALL1 EHD1 Rab11 -FIP1C Rab11 -FIP2 (6) (6) (6) (6) (10)(10) (10) (10) Ub level (α HA/ α Myc, % of WT) E B 250ー 500ー 1000ー 1500ー 2000ー 3000 750ー MK 1 2 1 2 1 2 RO WT RFFL KO 2500 (bp) Exon1 Exon2 RFFL (Genome)

5’-UTR gRNA ATG gRNA Primer (A) (B) (C) Exon1 RFFL KO (Genome) 1. Primer A-C: 2675bp 2. Primer B-C: 1627bp 1. Primer A-C: 1206bp 2. Primer B-C: 0bp C A 40 αRFFL WT KO (kDa) -RFFL

(50)

42

Figure 13. RFFL regulates ubiquitination of the Rab11 effectors in cell.

(A) Western blotting confirms the RFFL KO in 293MSR cells. (B) PCR analysis confirmed the RFFL KO. Schematic of the RFFL gene with the sgRNA-targeted sites, the start codon (ATG), and the PCR primers for genotyping are indicated. (C) RFFL KO in 293MSR cells was confirmed by DNA sequencing of the genomic locus. The guide sequences, PAM sequences, and the RFFL start codon are indicated. (D) Ubiquitination level of MB-Rab11-FIP1C in 293MSR (WT) and RFFL KO cells transfected with HA-Ub and RFFL-V5 or RFFL-H333A-V5 was measured by NA pull-down. (E) Ubiquitination level of the Rab11 effector proteins was quantified. At least 3 independent experiments were performed for

J

mock H333A-GFP A647-Tf merge A647-Tf merge H333A-GFP A647-Tf merge WT RFFL KO A647-Tf 2.5 h load (T-0 h) mock A647-Tf merge

A647-Tf 2.5 h load & 2 h chase (T-2 h)

mock A647-Tf merge A647-Tf merge H333A-GFP A647-Tf merge mock A647-Tf merge WT RFFL KO H333A-GFP

I

0 25 50 75 100 Intracellular Biotin-Tf (%) WT RFFL KO (9)(6) (9)(6) n.s. T-0 h T-4 h

(51)

43

quantification. Sample number was indicated in parentheses. Data represent means ± SE. *_{P<0.05. (F-H)}

Ubiquitination level of MB-MICALL1 (F), MB-EHD1 (G), and MB-Rab11-FIP2 (H) were measured as in panel D. (I) TfR recycling was measured in 293MSR (WT) and RFFL KO cells as the disappearance of internalized Biotin-Tf after 4 hours chase. (J) Fluorescent micrograph of internalized TfR in 293MSR (WT) or RFFL KO cells transfected with RFFL-H333A-GFP. TfR were labeled with A647-Tf for 2.5 hours at 37°C (T-0 h) and further chased for 2 hours (T-2 h).

ȕ3ș In vitro .@ RFFL .=@ Rab11-effector / Ub ä/ƪɦ RFFL KO *0 Rab11-effector / Ub ä.ŐˀD=6-'%˒RFFL-DN Ğǭ²Rab11-effector / Ub äDǐĸ!%+< RFFL + Rab11-effector Ǻ ´ǥ@+>˒ RFFL 0 Rab11-effector / Ub äDÖŘ@ôȻŞȶ >A%˓#*˒/ôȻŞDƪɦ@%:in vitro qPcÌƭťĬˋDɒ ++%˓7 ˒GST (glutathione-S-transferase) -EHD1˒GST-Rab11-FIP1C˒

ubiquitination enzymes˒DġȾɊ=?ȝɘ%34ːFig. 14Aˑ˓GST-EHD1 7%0

GST-Rab11-FIP1C D E1˒UbcH5c˒RFFL˒Ub +Ê.HPvgðŝ!

%Ŕ˒ĖɸDSacMq].)êʹ˒ŬUb Ŭ²Dǥ)IJ\at

deGSǀ.)ƪÐ%˓#/ȥƢ˒E1˒E2˒RFFL˒Ub /É)ý7A

14B and C lane 2ˑ˓MICALL1 =2 Rab11-FIP2 0ġȾɊ>/ȝɘčʺ*

'%%:˒ąˇȢȺ>/ȝɘDɨ8%˓ȝɘ% MB-MICALL1 7%0

MB-Rab11-FIP2 D E1˒UbcH5c˒RFFL˒HA-Ub +Ê.HPvgðŝŔ˒

ĖɸDNeutrAvidin FO\.)êʹ%˓Pull-down XuDĖɸ/ Ub ä

ƪÐ.ǥ˒RFFL / Ub äʟȠǄŞ/ƪÐ/%:, ǎ0Ŭ HA Ŭ²Dǥ% IJ\atdeGSǀ*/ƪɦ.ǥ>A%ːFig. 14Dˑ˓Rab11-FIP2 / Ub ä0 ubiquitination enzymes É)ý7A@Ɲ©*˒in cell .@ RFFL

ʕÛǳǡĬˋ+ùƮ.poly- and/or multiple-Ub äƪÐA%ːFig. 14E lane 3ˑ˓

MICALL1 / Ub ä0 ubiquitination enzymes É)ý7A@Ɲ©*˒poly- and/or multiple- and/or mono-Ub äƪÐA%ːFig. 14F lane 2ˑ˓MICALL1 / Ub ä0˒ E1 Dʱ+ĠĸƪÐA%ːFig. 14F lane 3ˑ˒UbcH5cːFig. 14F lane 4ˑ=

2HA-UbːFig. 14F lane 6ˑDʱ+ÉƪÐA-'%˓Ʌāǋ+.˒

(52)

44

/˒mono-Ub ä0ǉĢ-'%ːFig. 14F lane 5ˑ˓/ MICALL1 / RFFL ʽ

¸Ħǵ mono-Ub ä0˒ąˇȢȺ>/ȝɘʕȌ.)˒MICALL1 +ȥø

@ E3-ligase Ê.ȝɘA Ub äðŝDɺ%%:*@+ȶ>A@˓

/¥/E3 /ʬ0 RFFL D KO ); MICALL1 / Ub

ä.ŐˀDï6-'%++Ʉ)@˓ §/ȥƢ=?˒RFFL 0 Rab11-effetor Dǹź Ub

ä@ôȻŞȶ>A@˓

Figure 14. RFFL regulates ubiquitination of the Rab11 effectors in vitro.

(A) Recombinant GST-EHD1 or GST-Rab11-FIP1C was affinity purified and analyzed by SDS-PAGE with Coomassie Brilliant Blue (CBB) staining. Arrowheads indicate the full-length proteins. (B, C) In

vitro ubiquitination of GST-EHD1 (B), GST-Rab11-FIP1C (C) by RFFL was measured by Western

blotting after elution from the affinity beads. After the ubiquitination, total sample (B) or supernatant (C)

B 1 2 3 4 5 6 7 His-UBE1 His-UbcH5c His-RFFL Ub GST-EHD1 EHD1- Ub1 EHD1 EHD1- Ub1 EHD1 αUb (FK2) reprobe αGST (EHD1) GSH pull-down αGST (EHD1) reprobe αUb (FK2) To ta l EHD1 -Ub1 EHD1 -Ubn 70ー 55ー 210ー 140ー 90ー 70ー 55ー (kDa) 210ー 140ー 90ー 70ー 55ー 210ー 140ー 90ー 70ー 55ー C 1 2 3 4 5 6 7 His-UBE1 His-UbcH5c His-RFFL Ub GST-FIP1C αUb (FK2) reprobe αGST (FIP1C) GSH pull-down αUb (FK2) Sup (kDa) 210ー 140ー 90ー 70ー 210ー 140ー 90ー 70ー FIP1C FIP1C -Ub1 FIP1C -Ubn 210ー 140ー 90ー 70ー 55ー 40ー 35ー 20ー −FIP2 1 2 3 4 MB-Rab11-FIP2 His-UBE1/His-UbcH5c/HA-Ub His-RFFL 210ー 140ー 90ー 70ー (kDa) 90ー 70ー 210ー 140ー 90ー 70ー 55ー 40ー 35ー reprobe αHA (Ub) αMyc (FIP2) αHA (Ub) NA pull-down Sup FIP2 -Ubn E FIP1C- Ub A EHD1 FIP1C 140 90 70 55 40 (kDa) CBB staining GST- 1 2 3 4 5 6 7 His-UBE1 His-UbcH5c His-RFFL HA-Ub MB-MICALL1 F 210− 140− 210− 140− 210− 140− 90− 70− 55− 40− 35− αHA (Ub) αMyc (MICALL1) αHA (Ub) (kDa) −MICALL1 -Ub MICALL1 -Ubn −MICALL1 NA pull-down Sup D MB-MICALL1, Rab11-FIP2 on NA-beads His-E1, His-UbcH5c His-RFFL, HA-Ub, ATP

incubation 37°C, 2 h

Sup (auto-ubiquitination)

NA pull-down (Ub-MICALL1, Rab11-FIP2)

Analyzed with WB centrifugation

(53)

45

including E1, E2, RFFL, and Ub was analyzed for auto-ubiquitination to confirm their activity. (D) Schematic diagram of in vitro ubiquitination assay of MB-MICALL1 and MB-RAB11-FIP2 purified from mammalian cells. After the ubiquitination, supernatant (Sup) including E1, E2, RFFL, and HA-Ub is analyzed for auto-ubiquitination. The pellet containing MB-MICALL1 or MB-RAB11-FIP2 was washed, and their ubiquitination was analyzed by Western blotting after elution from NA-beads. (E, F) In

vitro ubiquitination of purified MB-Rab11-FIP2 (E) or MB-MICALL1 (F) from 293MSR cells by

(54)

46

ȕ

6ȓ ȶĲ

ƚǾȐ*0˒RE /ƱȻɯșDů Rab11-effector / Ub ä RFFL E3-ligase .

=')ÖŘA)@+DƑ>.%˓RFFL-DN Ğǭ²0˒R\aä

ERC DŎť˒ERC >/ȏɉ/XHRSDʮĮ%˓>.˒EE > LE ťǚäDʮĮ@+-˒Rab5 ʲŞ EE > Rab11 ʲŞ RE /ÑʹDʮĮ %˓ >.˒BioID ǀ.=?Ɗɞ RFFL ȥøČĥ+)˒Rab11-FIP1C˒-FIP2˒ -FIP5˒EHD1˒MICALL1 Dùī%˓RFFL-DN Ğǭ²0˒A>/ȥøČĥD

gduUb äDʮĮ@+*XHRSƱȻDǿȪ!%˓RFFL KO

0Rab11-FIP1C / Ub ä/8Dǐĸ!%˒in vitro .) RFFL 0ȥøČĥ

É)/Rab11-effector Dǹźǵ Ub ä@+ȃĆA%˓A>/ȥƢ=?˒ RFFL Dý9əƇ/ E3-ligase .=@ Rab11-effector / Ub ä˒XHRS Ȥɼ/ƱȻ.)ŜɛôƲ*@+ȃĆA%˓§/+Dɽ7 ƚȓ*0˒A7*ŗ>A)@Ǽɝ+˒£ċŦ Ķ)%Ǽɝ+DøC !@+*˒RFFL .=@ Rab11-effector Ub ä/ŢȳDȶĲ%˓ ȕ1ș RFFL-DN Ğǭ².@R\aä ERC ŎťÑĥƱƭ Ŧ /§Ù/ǾȐ.)˒RFFL 0 EE < LE .)ƭʏǭŁȿaoRɸ+ ʚŮǵ.Ǻ´ǥ Ub äD¤)``|Ñɠ3Ķ˒peripheral quality

control .ʬ@+ėÿA)@ 34˓ƋƚǾȐ*0˒HyVolution ɠơ

Dǥ@+*˒§Ù/ėÿ+Ʉ)˒EE < LE &*0- RE /ĹĐä

DƑ>. 53˒XHRSȤɼ.@ǤǢǵőÜȃĆA%˓7%

§Ù/ėÿ*0˒ƚǾȐ.)ǳɝ% RFFL-DN Ğǭ²/ɓǡē+ùƮ.˒

TfR, Rab5, Rab11 ƧĀʅ.Ɍȏ@+<˒TfR /XHRS/ʒň

RFFL-WT /ʕÛǳǡ.=?ȃA)% 53_{˓Ĭʴ.˒RFFL-WT ʕÛǳǡ.}

);R\aäERC ƒŭɟĲA%˓˒RFFL-WT + RFFL-DN Ğ

ǭ²/ǳǡʤùȖ/ʴ˒RFFL-DN Ğǭ²*/8R\aä ERC ɟĲA

%+>˒§Ù/ǾȐ.)RFFL-WT *ɟĲA%R\aä ERC 0

RFFL-WT /ʕÛʤ-ǳǡ.=@ Rab11-effector +/Ǻ´ǥ/ʨƗäíČ*

@+ȶ@˓Ǣǧ+)0˒ː1ˑù Ub ligase ǄŞäĞǭ²*;˒F{l

(55)

47

Ğǭ²*/8˒R\aä ERC ɟĲA%+,ː2ˑRFFL-WT =?;

RFFL-DN Ğǭ²/Ƌ Rab11-effctor +ōď.Ǻ´ǥ˒RE *gdu %+, ː3ˑCID ǀ.=? RFFL-WT + Rab11-effector /ōÖǵ-ȥø.=?R

\aäERC ŎťA%+>A@˓Ŗ')˒RFFL-WT + RFFL-DN

Ğǭ²/ǳǡʤùȖ/Ęø˒RFFL-WT 0 Rab11-effector +ȥøʎ<. Ub äDɒǽƒʫ*ɠʹ@/.ĳ)˒RFFL-DN Ğǭ²0 Rab11-effector D Ub ä@+* ˒ʨƗǵ-Ǻ´ǥŊɺA˒R\aäERC / ŎťDŊɺ)@+ȶ>A@˓ ȕ2ș RFFL-DN Ğǭ².@ȏɉXHRSÖŘ RFFL-DN Ğǭ²0ȏɉ/KhXHgZ\.0ŐˀDï6 ˒CME <

CIE .=?KhXHgZ\A%ȏɉDR\aä ERC .ɌȏXH

RSDʒň!%˓A>/ɓǡē0˒EHD1 KD (knockdown) 45,54_˒

MICALL1 KD 44˒class I Rab11-FIPs Ğǭ²55,56˒MYO5B tail 57/ɓǡē+ˇ®

)@˓Ŗ')˒RFFL-DN Ğǭ²0A>/ Rab11-effector /ƱȻDʮĮ@

ôȻŞ@˓Class I Rab11-FIPsːFIP1, 2, 5ˑ0 Rab11 +Ǻ´ǥ˒#A$A

XHRSȤɼ/ǭ-@u^\*˒ȿ/bHi{dR-ƭʏĞä.Ő

ˀDï6˒éɯǵ-Á.=?ȏɉ/XHRSDÖŘ)@ 58˓

Rab11-FIP2 0˒ERC > Rab11 ʲŞ/ RE DFRc/u\ƙȔ/Ƌû.ȋ

ã@+*ʃʊ@~aaoRɸ/˕(*@ MYO5B +éɯ)Á

575960˓7%˒EHD1 0 Rab11-FIP2 61_<_{MICALL1 +Ǻ´ǥ@+* ERC}

.ĹĐä˒ERC /ʃʊ< RE ȿ/ctä<ÑʹDɒ44,6263,64_˓A>/

Ǽɝ>˒RFFL-DN Ğǭ²+ Rab11-effector /ōÞ-ȥø0˒ERC > PM 3

/ȏɉ/XHRS=2 RE ȿ/ƭʏÖŘ.Ŝ˂- Rab11-effecor əø²

ːe.g., MYO5B-Rab11-Rab11-FIPs-EHD1ˑŎťDʮĮ)@ôȻŞȶ>A

@˓7%˒EHD1 < Rab11-FIP1C 0 EE/RE > TGN 3/ʃʊDÖŘ)?46,65˒

RFFL-DN Ğǭ²0 TGN46 DR\aä ERC 3+ǌĐ!%+<˒EHD1 <

Rab11-FIP1C / KD +ùƮ.65,66RFFL-DN Ğǭ² LE > TGN 3+ʃʊA

@ CI-M6PR DR\aä ERC 3+ǌĐ!%+>˒RFFL D¤%

Rab11-effector / Ub ä ERC > TGN 3/ʃʊ;ÖŘ)@ôȻŞȶ >A@˓

(56)

48

FRc<ŚķȘ¸Ħǵ-~aaoRɸ0Kh`|/ʃʊ. )ʽŁ.ʢɛ-őÜDƢ%˓BioID ǀ.=? RFFL /ȥøČĥ+) MYO1B (myosin IB) ˒MYO6 (myosin VI) ˒MYO1E (myosin IE) ˒KIF5B (kinesin family member 5B) ˒KIF16B (kinesin family member 16B) ùīA%ːFig.10Eˑ˓

MYO1B 0 TGN67₌₂_EE68_{>/ʃʊDÖŘ)?˒ȿ/XHRS}

.ʬ)@69˓MYO1E 0 ERC 3/ TfR /ʃʊ.ʬ)@ 70˓MYO6

0Ub binding h}HDƕ71,72˒KD @+ Rab5 DƧĀʅ.ʷȏ!73˒EE

>ERC 3/ȏɉ/ʃʊDʮĮ@74_˓_{KIF5B 0 EE > RE 3/ʃʊDÖŘ}

)?75˒RFFL-DN Ğǭ²*ɟĲA@=- LY /ƧĀʅ3/ĹĐäDɬĶ

@76˓A>/Ǽɝ=?˒RFFL 0XHRSȤɼ.)A>/~

aaoRɸ+Rab11-effctor /Ǻ´ǥDÖŘ@ôȻŞ;ȶ>A@˓

ȕ3ș RFFL-DN Ğǭ².@ EE > RE 3/ÑʹÖŘ Ʌāǋ+.˒RFFL-DN Ğǭ²0 Rab5 ʲŞ EE > Rab7 ʲŞ LE /Ñʹ DʮĮ@+-˒Rab5 ʲŞ EE > Rab11 ʲŞ RE /ÑʹDʮĮ%˓ /ɓǡē+Ʉ)˒RFFL-DN Ğǭ²0 EGFR =2 Dextran / LY ʃʊDʮ Į@+-˒ȏɉ/XHR/8DʮĮ%˓LY 3+ʃʊ!@ȏɉa o R ɸ / * ˒RFFL-DN Ğ ǭ ² . = ' ) ʃ ʊ ʮ Į A @ ȏ ɉ 0

rΔF508-CFTR /8*'%˓RFFL 0 peripheral quality control .)ƭʏǭŁ

ȿaoRɸ*@rΔF508-CFTR+ʚŮǵ.Ǻ´ǥ@%:34˒RFFL-DN Ğ

ǭ²0EE/ERC .)ƭʏǭŁȿaoRɸDgdu˒LY ʃʊDʒň

!@ôȻŞȶ>A@˓RFFL-DN Ğǭ².=@ Rab5 ʲŞ EE > Rab11 ʲŞ RE 3/ÑʹʮĮ0#>˒Rab11-effector /ƱȻʮĮíČ*@+ȶ>A @˓³ƅ->˒Rab11-FIP2 0 RE /ÖŘ&*0-˒EE < RE /ÓƗƷʳ. ;ʬ@ėÿ<77˒EHD1 < MICALL1 0˒EE > RE 3/ȏɉʃʊ.@

ctǝ/ȿŎť.ʬ)?44,78˒EHD1 =2 MICALL1 KD .=? EE

+ ERC ǌĐ)7ȏɉ/ʃʊʮĮA@ėÿ@%:*@ 78_˓

RFFL-DN Ğǭ²;ùƮ. EE D ERC .ǌĐ!@+>˒RFFL =2¥/ E3-ligase .=@ EHD1 =2 MICALL1 / Ub äÖŘ0˒EE > RE 3/c

ユビキチンリガーゼRFFLによるエンドソーム機能制御機構の解明