Ani ASRYQKNARTKACCLLIILAVILAIIILAAVL Ang ASRYQKNARNKACCLLVILAVILTIIVLAATL Fox AARYQKAARNKSCCLLLILAVILTIVILAIVL Ncr AARYQKNARGKACCLLLILSVILTIVLLAVFL Mor AARYQKNARSKACCLLLILSVILTIVLLAIFL Pbr ASRYQKKARNKACCLLLILAVVLVIIVLAATL Cim ASRYQKNARNKACCLLLILAIVLVIIVLAVTL

Bci AHEYQRRAGRRAACLLLILSVVVAVVLLAIL- Sce AMRYQKRTSRWRVYLLIVLLVMLLFIFLIM-- Cal ANEYQKQKGKWSCILLVALCIFLLVIVLIVV- Spo ANEHSRKARKRSFCFLVILVVILGVILTALIM

wild type I257D C253,254S I257DC253,254S

10 mm

nucnuc

Ile257Asp

wild type

Cys253,254Ser Ile257Asp

Cys253,254Ser

10 mm

A

C

D

Cys253Cys254

Ile257

24 s

ec

10 mm

wild type

E

Cys253,254Ser

Ile257Asp

Cys253,254Ser Ile257Asp

TMD

*

wild

typ

e

I257D

C253,2

54S

I257D

C253,2

54S

GFPPepAs

PepA

WB a-PepA

wild

typ

e

I257D

C253,2

54S

I257D

C253,2

54S

WB a-GFP

actin

B

10 mm

18 s

ec

GFP-TlgBj

k

( ((

'

'

'

'

'

'

TlgB

RabA

56 s

ec

10 mm

5 mm

A

B

MCA MMA

30ºC

37ºC

42ºC

wt tlgBΔtlgAΔtlgB ΔtlgAΔ wt tlgBΔ

tlgAΔtlgB ΔtlgAΔ

WT tlgAD tlgBD tlgAD�tlgBD

0

5

10

15

20

25

30

WT tlgAD tlgBDtlgAD�tlgBD

inte

gra

ted in

tensi

ty 1

0e-6

arb

itrary

units

A

B

C***

***n.s.

B

A

HA3-Vti1

Qb

I IP FT IP FT

α-Stx α-Pre

HA3-Tlg1

HA3-Syn8

HA3-Vam7

Qc

HA3-SynA

Ykt6-HA3

Nyv1-HA3

R

I IP FT IP FT I IP FT IP FT

α-Stx α-Pre α-Stx α-Pre

SedV TlgB PepA

SedV TlgB PepA

SedV TlgB PepA

I IP FT IP FT

α-Stx α-PreI IP FT IP FT I IP FT IP FT

α-Stx α-Pre α-Stx α-Pre

I IP FT IP FT

α-Stx α-PreI IP FT IP FT I IP FT IP FT

α-Stx α-Pre α-Stx α-Pre

I IP FT

α-SedV α-TlgB α-PepA

GFP-SedV

GFP-TlgB

IP FT IP FT

GFP-PepA

blot -GFPα

Vti1

Tlg

1

Syn

8

SedV

TlgB

PepA

Vam

7

Syn

A

Ykt

6

Nyv1C

wt tlgBΔvps45Δ pepAΔ

pepAΔ pepAΔsltAΔ

vps45Δ vps45ΔsltAΔ

A

B

In PD FT PD FT

a-TlgB pre a-PepAIn PD FT PD FT

pre

1 2 3 4 5 6 7 8 9 10

Vps33

In PD FT PD FT

a-TlgB pre a-PepAIn PD FT PD FT

pre

Vps45

IPs of Vps45-HA3 extracts

IPs of Vps33-HA3 extractsIn

put T

NT

IP,

pre

IP-T

lgB

, a

Input T

NT

IP,

pre

IP-P

ep

A, a

TNT Vps33-HA3 TNT Vps33-HA3

TNT Vps45-HA3 TNT Vps45-HA3

Vps45

Vps33

SM1

AB

Direct or indirect interaction test

extracts

1 2 3 4 5 6 7 8 9 10

AB

SM1

HA3 Stx1

Direct interaction test

IP IP

A B

IPs of Vps45-HA3 extracts IPs of TNT-synthesized proteins

SM1

SM2

SM1HA3

Stx2Stx1

SM1

SM1

AB

Stx1

Stx1

or

Stx1

Stx1

I IP FT IP FT

α-Pep12 α-Pre

Vps41-HA

Vps8-HA

16

1118

338

3

16

1118

3341

39

LE/MVB

EE

vac

Rab5Rab7

CORVET HOPS

TlgB

Vti1

Vam7

Syn8

TlgATlgA SynA

Nyv1

Ykt6

SedV

PepA

Qa Qb Qc R

TG

N/P

GE

Go

lgi

LE

<<

EE

VA

C

A

SedV

Vti1 (Qb)Syn8/Tlg1(Qc)SynA/Ykt6 (R)

Nyv1 ?

exocytosis

earlyGolgi

EE

V

PGE

Nyv1 ?

earlyGolgi

V

TGN

Nyv1 ?

Vti1 (Qb)Vam7 (Qc)Nyv1 (R)

earlyGolgi

LE

Vti1 (Qb)TlgA (Qc)SynA (R)

Nyv1 ?

endocytosis

earlyGolgi

Vac

TlgB

TGN

TlgB

Pep12PepA

Pep12PepA

Pep12PepA

B

Vti1 (Qb)Syn8/Tlg1(Qc)SynA/Ykt6 (R)

?