Btn1 regulates endosome–Golgi retrograde transport • Kama et al.

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JCBSupplemental material

Kama et al., http://www.jcb.org/cgi/content/full/jcb.201102115/DC1

Figure S1.  The deletion or overexpression of BTN1 does not alter localization of a wide variety of endo-some-trafficked  proteins. (A) The localization of many endosome-trafficked proteins is not altered by the deletion or overexpression of BTN1. WT, btn1, and WT cells overexpressing BTN1 from a multicopy plasmid were transformed with plasmids expressing GFP-tagged Snx4, Vps27, Vps10, Tlg1, Tlg2, CPY, Snc1, Fur4, and Ste2. Cells were grown to midlog phase at 26°C and examined by confocal micros-copy. Light indicates the DIC panel. Bars, 1 µm. (B) CPY is not secreted by btn1 cells. WT, btn1, vps10, and vps26 cells were grown to midlog phase at 26°C, diluted serially (×10), and plated by drops onto nitrocellulose filters placed on rich me-dium plates. After 48 h at 26°C, the filters were re-moved, washed, and probed with anti-CPY antibodies using chemiluminescent detection. (C) GFP-Yif1 ap-pears more strongly degraded in btn2 than in btn1 cells. WT, btn1, btn2, and yck3 cells were grown to midlog phase at 26°C before pro-cessing for Western analysis using anti-GFP to detect GFP-Yif1 and free GFP degradation products and anti-actin as a loading control. Arrowheads indicate the localization of GFP-Yif1 as well as free GFP, which accumulates upon degradation in the vacuole. Note the prominent band of free GFP in btn2 cells. (D) The deletion of BTN1, BTN2, or YCK3 reduces the secretion of active -mating factor. WT (BY4742) and MAT- btn1, btn2, and yck3 cells were grown to midlog phase at 26°C, and equal OD600 amounts of cells were spotted onto a MATa sst2 tester lawn. Cells were grown for 2 d at 26°C before photodocumentation (note that the dark background illustrates the halo of arrested cells).

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Figure S2.  The deletion and overexpression of BTN1 have opposing effects on the growth of temperature-sensitive mutants of Golgi SNARE proteins. (A) The overexpression of BTN1 partially inhibits the growth of vti1 mutants. vti1-1, vti1-2, and vti1-11 cells were transformed with a control vector or multicopy plasmids overexpressing BTN1 (+BTN1) or VTI1 (+VTI1). Cells were grown to midlog phase at 26°C on synthetic medium, diluted serially (×10), and plated by drops onto preheated solid medium. Cells were grown at the indicated temperatures for 48 h before photodocumentation. (B) The overexpression or de-letion of BTN1 has opposing effects on the growth of ykt6-1 cells. (top) ykt6-1 cells transformed with a control plasmid, a plasmid overexpressing BTN1 (+BTN1), or a plasmid overexpressing YKT6 (+YKT6) were grown to midlog phase at 26°C on synthetic medium, diluted serially (×10), and plated by drops onto prewarmed solid medium. (bottom) In parallel, ykt6-1 cells transformed with a control plasmid and ykt6-1 GAL-BTN1 cells transformed with either a control plasmid or a plasmid overexpressing BTN1 (+BTN1) were grown to midlog phase on galactose-containing synthetic medium and were shifted to glucose-containing synthetic medium for 16 h (to yield the btn1 condition). Cells were plated and grown in the same manner as previously described. (C) The overexpression of BTN1 partially inhibits the growth of sed5-1 cells. sed5-1 cells were transformed with a control vector or multicopy plasmids overex-pressing BTN1 (+BTN1) or SED5 (+SED5). Cells were grown to midlog phase at 26°C on synthetic medium, diluted serially (×10), and plated by drops onto preheated solid medium. Cells were grown at the indicated temperatures for 48 h before photodocumentation. (D) The overexpression of BTN1 has lit-tle to no effect on the growth of sec21-2, sec22-2, sec1-1, and sec9-4 cells. sec21-2, sec22-2, sec1-1, and sec9-4 cells were transformed with a control plasmid, a multicopy plasmid expressing BTN1 (+BTN1), or their respective WT copies of the mutated gene (as indicated, except in the case of sec9-4 cells, wherein SSO1 is used to rescue the mutation). Cells were grown to midlog phase at 26°C on synthetic medium, diluted serially (×10), and plated by drops onto preheated solid medium. Cells were grown at the indicated temperatures for 48 h before photodocumentation. (E) The overexpression or dele-tion of BTN1 has no effect on the growth of WT cells. WT cells transformed either with a control vector (WT) or a multicopy plasmid overexpressing BTN1 (+BTN1) were grown along with btn1 cells to midlog phase at 26°C on synthetic medium, diluted serially (×10), and plated by drops onto preheated solid medium. Cells were grown at the indicated temperatures for 48 h before photodocumentation.

Btn1 regulates endosome–Golgi retrograde transport • Kama et al. S3

Figure S3.  The deletion of BTN1 does not affect AP3-mediated protein sorting nor localization of an AP-3 component. (A) The deletion of BTN1 does not affect AP3-mediated protein sorting. WT, btn1, yck3, and apl5 cells were transformed with a plasmid expressing a Nyv1-Snc1 fusion protein. Cells were grown to midlog phase at 26°C on synthetic medium, pulse-chase labeled with FM4-64, and examined by confocal microscopy. Note the lo-calization of Nyv1-Snc1 to the plasma membrane in cells defective in AP3 transport (i.e., yck3 and apl5 cells). (B) Apl5 is not mislocalized in cells lacking BTN1. WT and btn1 cells expressing APL5-GFP from its chromosomal locus were grown to midlog phase at 26°C on synthetic medium, pulse-chase labeled with FM4-64, and examined by confocal microscopy. (A and B) Merge indicates merger of the GFP and FM4-64 panels. Light indicates the DIC panel. Bars, 1 µm.

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Figure S4.  Yck3 is not mislocalized in btn1 cells, although YCK3 overexpression inhibits the growth of Golgi SNARE mutants. (A) The deletion of BTN1 does not mislocalize GFP-Yck3. WT and btn1 cells expressing GFP-YCK3 from the YCK3 chromosomal locus were grown to midlog phase at 26°C on synthetic medium, pulse-chase labeled with FM4-64, and examined by confocal microscopy. Merge indicates merger of the GFP and FM4-64 panels. Light indicates the DIC panel. Note the localization to vacuolar-limiting membrane and plasma membrane in both cell types. Bars, 1 µm. (B) Overexpression of YCK3 inhibits the growth of sed5-1 cells. sed5-1 cells transformed with a control plasmid or multicopy plasmids expressing YCK3, BTN1, HRR25, or SED5 (+YCK3, BTN1, HRR25, or SED5, respectively) were grown to midlog phase at 26°C on synthetic medium, diluted serially (×10), and plated onto pre-warmed solid synthetic selective medium. Plates were incubated for 48 h before photodocumentation. (C) Overexpression of YCK3 or BTN1 inhibits the growth of ykt6-1 cells. ykt6-1 cells transformed with a control plasmid or multicopy plasmids expressing YCK3, BTN1, or YKT6 (+YCK3, BTN1, or YKT6, re-spectively) were grown to midlog phase at 26°C on synthetic medium, diluted serially (×10), and plated onto prewarmed solid synthetic selective medium. Plates were incubated for 48 h before photodocumentation.

Btn1 regulates endosome–Golgi retrograde transport • Kama et al. S5

Figure S5.  The deletion of BTN2 also affects Sed5 phosphorylation and Golgi morphology. (A) Sed5 is underphosphorylated in cells lacking BTN2. WT cells, btn1, and btn2 cells were grown to midlog phase on synthetic medium at 26°C. Cells were harvested and lysed, and samples of the postnuclear TCL were subjected to SDS-PAGE on an 11.5% polyacrylamide gel. Blots were probed with anti-Sso antibodies to detect Sso1/2 as a loading control, whereas anti-Sed5 antibodies were used to detect Sed5. NP indicates the lower mass/NP form of Sed5, whereas P indicates the higher mass/P form. The histogram shown is the quantification of the upper (P) and lower (NP) bands of Sed5 after normalization for Sso loading. Note the presence of mainly the lower molecular mass (i.e., under/NP) form of Sed5 (NP) in both the btn1 and btn2 cells. The data shown are representative of multiple replicates of the experiment (n = 3). (B, top) The deletion of BTN2 enhances Golgi clustering in cells expressing GFP-Sed5. WT, btn2, and WT cells overexpressing BTN2 from a multicopy plasmid (BTN2) were transformed with a single-copy plasmid expressing GFP-Sed5, grown to midlog phase at 26°C, and examined by confocal microscopy. Note the larger GFP-Sed5–labeled puncta in the btn2 cells. (bottom) The overexpression of BTN2 does not inhibit Golgi clustering in cells expressing GFP-Sed5317A. WT, btn2, and WT cells overexpressing BTN2 from a multicopy plasmid (BTN2) were transformed with a single-copy plasmid expressing GFP-Sed5317A, grown to midlog phase, and examined by confocal microscopy. Note the large puncta observed in all cell types, even in cells overexpressing BTN2. Light indicates the DIC panel. Bars, 1 µm.

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Table S1. Localization of GFP-Yif1 in btn1 and yck3 cells

Strain GFP-Yif1 localization

Golgi Golgi + endosome Endosome Vacuole

% % % %WT 85 ± 1.7 6 ± 2 2.3 ± 0.6 6.7 ± 3.1btn1a 18 ± 2 40.3 ± 1.5 25.3 ± 2.1 16.4 ± 1.5btn2 13.3 ± 1.5 7 ± 1.7 47.3 ± 1.5 32.4 ± 3.1btn1CLN3 71.3 ± 1.2 11.7 ± 1.2 13 ± 2.6 4 ± 1.7yck3 16.3 ± 4.9 42 ± 1 27 ± 5.3 14.7 ± 1.2btn1yck3 23 ± 2.5 24 ± 1 43 ± 1.5 10 ± 1

GFP-Yif1 localization is based on counting 100 cells. n = 3 experiments, and the average ± SD is given.aObtained from the European Saccharomyces Cerevisiae Archive for Functional Analysis.

Table S2. Localization of Kex2-GFP in btn1, yck3, and DHHC 5× cells

Strain Kex2-GFP localization

Golgi LE

% %WT 79.7 ± 4.7 20.3 ± 4.7btn1 43.7 ± 3.1 56.3 ± 3.1btn1CLN3 77.7 ± 2.5 22.3 ± 2.5yck3 47.7 ± 2.5 52.3 ± 2.5DHHC 5× 49.3 ± 1.5 50.7 ± 7

Kex2-GFP localization is based on counting 100 cells. n = 3 experiments, and the average ± SD is given.

Table S3. Localization of GFP-Sed5 in WT cells lacking or overexpressing BTN1

Strain GFP-Sed5 localization

Dispersed Golgi Clustered Golgi

% %WT (GFP-SED5) 86 ± 2.7 14 ± 2.7WT (GFP-SED5) BTN1 94.7 ± 1.5 5.3 ± 1.5WT (GFP-SED5) btn1 64.7 ± 2.5 35.3 ± 2.5WT (GFP-SED5317A) 59 ± 4.4 41 ± 4.4WT (GFP-SED5317A) BTN1 83.3 ± 4 16.7 ± 4WT (GFP-SED5317A) btn1 56.3 ± 6.1 43.7 ± 6.1WT (GFP-SED5317D) 85.3 ± 2.1 14.7 ± 2.1WT (GFP-SED5317D) BTN1 91.3 ± 2.5 8.7 ± 2.5WT (GFP-SED5317D) btn1 64 ± 2.7 36 ± 2.7

GFP-Sed5 localization is based on counting 100 cells. n = 3 experiments, and the average ± SD is given.

Table S4. Localization of GFP-Sed5 in yck3 cells

Strain GFP-Sed5 localization

Dispersed Golgi Clustered Golgi

% %WT 87 ± 2 13 ± 2btn1 63 ± 3.5 37 ± 3yck3 58 ± 3 42 ± 3

GFP-Sed5 localization is based on counting 100 cells. n = 3 experiments, and the average ± SD is given.

Btn1 regulates endosome–Golgi retrograde transport • Kama et al. S7

Table S5. Localization of GFP-Yif1 in cells expressing mutant Yck3 proteins or lacking DHHC palmitoyl transferases

Strain GFP-Yif1 localization

Golgi Golgi + endosome + vacuole Vacuole

% % %WT 84.7 ± 1.5 10.7 ± 1.5 4.6 ± 1.5DHHC 5× 8.3 ± 0.6 36.7 ± 5 55 ± 5YCK3-FYVE 9.3 ± 1.5 45.7 ± 3.1 45 ± 2.7YCK3-FYVE btn1 30 ± 7 56 ± 3.6 14 ± 3.5YCK3-FYVE BTN1 13 ± 2 41 ± 1.7 46 ± 1btn1a 36.7 ± 1.5 57.7 ± 2.1 5.7 ± 0.6YCK3-VAM3 btn1 48 ± 2.7b 39 ± 2c 13 ± 3d

GFP-Yif1 localization is based on counting 100 cells. n = 3 experiments, and the average ± SD is given.aStrain background = RKY11.bP = 0.02; significance between YCK3-VAM3 btn1 and btn1.cP = 0.007; significance between YCK3-VAM3 btn1 and btn1.dP = 0.027; significance between YCK3-VAM3 btn1 and btn1.

Table S6. Localization of GFP-Sed5 in cells lacking or overexpressing BTN2

Strain GFP-Sed5 localization

Dispersed Golgi Clustered Golgi

% %WT (GFP-SED5) 90.7 ± 0.6 9.3 ± 0.6WT (GFP-SED5) BTN2 90 ± 1.0 10 ± 1.0btn1 (GFP-SED5) 69.7 ± 6.5 30.3 ± 6.5btn2 (GFP-SED5) 66 ± 8.7 34 ± 8.7WT (GFP-SED5317A) 66.3 ± 1.2 33.7 ± 1.2WT (GFP-SED5317A) BTN2 70 ± 2.0 30 ± 2.0btn1 (GFP-SED5317A) 58.3 ± 2.5 41.7 ± 2.5btn2 (GFP-SED5317A) 68 ± 3.0 32 ± 3.0

GFP-Sed5 localization is based on counting 100 cells. n = 3 experiments, and the average ± SD is given.

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Table S7. Yeast strains used in this study

Strain Genotype Source

BY4741 MATa his31 leu2 met150 ura30 EUROSCARFBY4742 MAT- his31 leu2 met150 ura30 EUROSCARFW3031a-SEC7RFP MATa can1 his3 leu2 lys2 ura3 ade2 trp1::TRP1::TPI1-DsRed-SEC7 Gerst laboratoryRKY8 MATa leu2 met150 ura30 GAL1-GFP-BTN1::HIS3 This studyRKY9 MATa leu2 met150 ura30 GAL1-GFP-YCK3::HIS3 This studyRKY10 MATa his31 leu2 met150 ura30 btn1::kanMX yck3::natMX This studyRK1 MATa ura3 leu2 trp1 ade8 sed5::GFP-SED5::his5 + can1 Gerst laboratoryRK1 btn1 MATa ura3 leu2 trp1 ade8 sed5::GFP-SED5::his5 + can1 btn1::natMX This studyRK2 MATa ura3 leu2 trp1 ade8 sed5::GFP-SED5317A::his5 + can1 Gerst laboratoryRK2 btn1 MATa ura3 leu2 trp1 ade8 sed5::GFP-SED5317A::his5 + can1 btn1::natMX This studyRK3 MATa ura3 leu2 trp1 ade8 sed5::GFP-SED5317D::his5 + can1 Gerst laboratoryRK3 btn1 MATa ura3 leu2trp1 ade8 sed5::GFP-SED5317D::his5 + can1 btn1::natMX This studyFvMY7 MATa leu2-3,112 ura3-52 his3-200 ade2-101 trp1-901 suc2-9 mel-vti1-1 G. Fischer von MollardFvMY21 MATa leu2-3,112 ura3-52 his3-200 ade2-101 trp1-901 suc2-9 mel-vti1-11 G. Fischer von MollardFvMY24 MATa leu2-3,112 ura3-52 his3-200 ade2-101 trp1-901 suc2-9 mel-vti1-2 G. Fischer von MollardSARY 166 MAT- ykt6::LEU2 (CEN6, TRP1, ykt6-1) D. BanfieldSARY166-RK MAT- ykt6::LEU2 GAL1-GFP-BTN1::HIS3 (CEN6, TRP1, ykt6-1) This studySF750-14D– MAT- ura3-52 leu2-3,112 his4-580 s1-1 R. Schekmana

NY782 MATa ura3-52 leu2-3,112 s9-4 P. Novickb

RSY1309 MATa ura3-52 leu2-3,112 lys2-801 suc2-9 s21-2 A. Spangc

RSY324 MATa ura3-52 s22-2 R. SchekmanCBY265 MATa trp1 ura3 leu2 lys2 sed5-1 C. Barlowebtn1 MATa his31 leu2 met150 ura30 btn1::kanMX EUROSCARFbtn1- MAT- his31 leu2 met150 ura30 btn1::kanMX EUROSCARFRKY14 (btn1-) MAT- his31 leu2 met150 ura30 btn1::natMX This studybtn2 MATa his31 leu2 met150 ura30 btn2::kanMX EUROSCARFbtn2- MAT- his31 leu2 met150 ura30 btn2::kanMX EUROSCARFsst2 MATa his31 leu2met150 ura30 sst2::kanMX EUROSCARFvps10 MATa his31 leu2 met150 ura30 vps10::kanMX EUROSCARFvps26 MATa his31 leu2 met150 ura30 vps26::kanMX EUROSCARFyck3 MATa his31 leu2 met150 ura30 yck3::kanMX EUROSCARFyck3- MAT- his31 leu2 met150 ura30 yck3::kanMX EUROSCARFsed5 MATa ura3::GAL-SED5 sed5::LEU2 trp1-1 J. Gerst3866-BJ (APL5-GFP) MATa pep4::HIS3 prb-1.6R lys2-208 trp1101 ura3-52 gal2

APL5::GFP::TRP1C. Ungermann

2052-NDY1690 (akr1 ark2 pfa3 pfa4 pfa5 YCK2-CCIISS)

MATa ura3 his3 leu2 akr1 ark2 pfa3 pfa4 pfa5::G418R lys2::YCK2(CCIIS)

C. Ungermann

522-BJ (NOP1pr-VPS41) MATa pep5::HIS3 prb-1.6R HIS3 lys2-208 trp1101 ura3-52 gal2 can vps41::KANMx6 ura3::pRS406-NOP1pr-VPS41

C. Ungermann

RKY11 (btn1 in 522BJ) MATa pep5::HIS3 prb-1.6R HIS3 lys2-208 trp1101 ura3-52 gal2 can vps41::KANMx6 ura3::pRS406-NOP1pr-VPS41 btn1::NAT1

This study

1322-BJ (yck3::cys-VAM3-TMD) MATa pep5::HIS3 prb-1.6R HIS3 lys2-208 trp1101 ura3-52 gal2 can vps41::KANMx6 ura3::pRS406-NOP1pr-VPS41 yck3::cys-VAM3-TMD-hyphNT1

C. Ungermann

RKY12 (yck3::cys-VAM3-TMD btn1)

MATa pep5::HIS3 prb-1.6R HIS3 lys2-208 trp1101 ura3-52 gal2 can vps41::KANMx6 ura3::pRS406-NOP1pr-VPS41 yck3::cys-VAM3-TMD-hyphNT1 btn1::NAT1

This study

4429-BT3505 (yck3::cys-FYVE) MATa pep4::HIS3 prb-1.6R lys2-208 trp1101 ura3-52 gal2:: YCK3cys::FYVE-TRP1

C. Ungermann

RKY13 (yck3::cys-FYVE btn1) MATa pep4::HIS3 prb-1.6R lys2-208 trp1101 ura3-52 gal2:: YCK3cys::FYVE-TRP1 btn1::KAN

This study

RKY15 MATa his31 leu2 met150 ura30 GFP-BTN1 3UTR::natMX This study

EUROSCARF, European Saccharomyces Cerevisiae Archive for Functional Analysis.aUniversity of California, Berkeley, Berkeley, CA.bUniversity of California, San Diego, La Jolla, CA.cBiozentrum, University of Basel, Basel, Switzerland.

Btn1 regulates endosome–Golgi retrograde transport • Kama et al. S9

Table S8. Plasmids used for this study

Plasmid name Gene expressed/deleted

Promoter/ terminator

Vector Cloning sites Type Selectable marker

Source

pRS316-GFP-YIF1 HA-GFP-YIF1 ADH1/ADH1 pRS316 BamHI CEN URA3 Gerst laboratoryp313-RFP-YIF1 HA-RFP-YIF1 ADH1/ADH1 pAD313 SalI–SacI CEN HIS3 This studypAD54-CLN3 HA-CLN3 ADH1/ADH1 pAD54 SalI–SacI 2µ LEU2 This studypRS313-BTN2-mRFP Myc-BTN2-mRFP

(w/o SacI)ADH1/ADH1 pRS313 BamHI CEN HIS3 Gerst laboratory

pRS426-BTN2-GFP Myc-BTN2-GFP ADH1/ADH1 pRS426 BamHI 2µ URA3 Gerst laboratorypUG23-KEX2-GFP KEX2-GFP ADH1/ADH1 pUG23 SpeI–SalI CEN HIS3 This studypUG46-GFP-BTN1(+3UTR) GFP-BTN1 ADH1/ADH1 pUG46 EcoRI–SalI CEN URA3 This studypAD54-BTN1(+3UTR) HA-BTN1 ADH1/ADH1 pAD54 SalI–SacI 2µ LEU2 This studypAD54-GFP-BTN1(+3UTR) GFP ADH1/ADH1 pAD54-BTN1 SalI 2µ LEU2 This studypAD54-RFP-BTN1(+3UTR) RFP ADH1/ADH1 pAD54-BTN1 SalI 2µ LEU2 This studypRS426-BTN1(+3UTR) Myc-BTN1 ADH1/ADH1 pRS426

(w/o SalI–SacI)SalI–SacI 2µ URA3 This study

pRS426-GFP-BTN1(+3UTR) GFP ADH1/ADH1 pRS426-BTN1 (w/o SalI–SacI)

SalI 2µ URA3 This study

pRS426-YKT6(+3UTR) Myc-YKT6 ADHI/ADHI pRS426 (w/o SalI–SacI)

SalI–SacI 2µ URA3 This study

pRS313-ykt6-1(+3UTR)a Myc-ykt6-1 ADH1/ADH1 pRS313 (w/o SalI–SacI)

SalI–SacI CEN HIS3 This study

pAD54-SED5 HA-SED5 ADH1/ADH1 pRS315 SalI–SacI 2µ LEU2 Gerst laboratorypRS315-SED5 HA-SED5 ADH1/ADH1 pRS315 SalI–SacI CEN LEU2 Gerst laboratorypRS315-SED5317A HA-SED5 ADH1/ADH1 pRS315 SalI–SacI CEN LEU2 Gerst laboratorypRS315-SED5317D HA-SED5 ADH1/ADH1 pRS315 SalI–SacI CEN LEU2 Gerst laboratorypRS315-GFP-SED5 HA-SED5 ADH1/ADH1 pRS315 SalI–SacI CEN LEU2 Gerst laboratorypRS316-GFP-SED5 HA-SED5 ADH1/ADH1 pRS316

(w/o SalI–SacI)SalI–SacI CEN URA3 Gerst laboratory

pAD54-RFP-VPS27 RFP-VPS27 ADH1/ADH1 pAD54 SalI–SacI 2µ LEU2 Gerst laboratorypRS315-GFP-cSNC1 GFP-cSNC1 ADH1/ADH1 pRS315 BamHI CEN LEU2 Gerst laboratorypRS315-GFP-TLG1 GFP-TLG1 ADH1/ADH1 pRS315 BamHI CEN LEU2 Gerst laboratorypRS315-GFP-TLG2 GFP-TLG2 ADH1/ADH1 pRS315 BamHI CEN LEU2 Gerst laboratorypGO426-GFP-VPS27 GFP-VPS27 CPY1 pRS426 BamHI 2µ URA3 S. EmrpAD54-GFP-SNX4 HA-GFP-SNX4 ADH1/ADH1 pAD54 SalI 2µ LEU2 Gerst laboratorypAD54-VPS10-GFP HA-VPS10-GFP ADH1/ADH1 pAD54 SalI–SacI 2µ LEU2 Gerst laboratorypRS316-FUR4-GFP HA-FUR4-GFP ADH1/ADH1 pRS316 BamHI CEN URA3 Gerst laboratorypGALBglII-CPY

(1–50)GFPCPY1–50-GFP GAL1 pGALBglII CEN URA3 O. Delocheb

pRS315-STE2-GFP STE2-GFP ADH1/ADH1 pRS315 CEN LEU2 R. Piperc

pAD54-VTI1 HA-VTI1 ADH1/ADH1 pAD54 SalI–SacI 2µ LEU2 Gerst laboratorypADH-SEC21 Myc-SEC21 ADH1/ADH1 pAD6 SalI–SacI 2µ LEU2 Gerst laboratorypADH-SEC22 Myc-SEC21 ADH1/ADH1 pAD6 SalI–SacI 2µ LEU2 Gerst laboratorypRS426-SSO1 SSO1 ADH1/ADH1 pRS426 SalI–SacI 2µ URA3 Gerst laboratorypAD54-SEC1 HA-SEC1 ADH1/ADH1 pAD54 SalI–SacI 2µ LEU2 Gerst laboratorypAD54-YCK3(+3UTR) HA-YCK3 ADH1/ADH1 pAD54 SalI–SacI 2µ LEU2 This studypAD54-GFP-YCK3(+3UTR) GFP ADH1/ADH1 pAD54-HA-YCK3 SalI–SacI 2µ LEU2 This studypUG46-GFP-YCK3(+3UTR) GFP-YCK3 ADH1/ADH1 pUG46 SmaI–SalI CEN URA3 This studypGNS416-NYV1-SNC1 GFP-NYV1-SNC1 PGNS416 CEN URA3 C. UngermannpRS425-HRR25 HRR25 ADH1/ADH1 pRS425 SalI–SacI 2µ LEU2 This study

w/o, without.aSee Materials and methods for details on construction.bUniversity of Geneva, Geneva, Switzerland.cUniversity of Iowa, Iowa City, IA.

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Table S9. Glossary of genes used in this study

Gene name Systematic name Protein name Protein description Cellular localization

AKR1 YDR264C Akr1 Palmitoyl transferase involved in protein palmitoylation GolgiAPL5 YPL195W Apl5 -Adaptin–like subunit of the clathrin-associated protein

complex (AP-3); functions in direct Golgi–vacuole transportGolgi

BET1 YIL004C Bet1 SNARE required for vesicular transport between the ER and Golgi complex

ER

BOS1 YRL078C Bos1 SNARE required for vesicular transport between the ER and Golgi complex

ER

BTN1 YJL059W Btn1 Orthologue of human CLN3; originally proposed to regulate vacuolar pH homeostasis

trans-Golgi, vacuole (upon overexpression)

BTN2 YGR142W Btn2 Snc1 SNARE- and retromer-binding protein that facilitates both LE–Golgi protein retrieval and prion curing

LE, insoluble protein deposit compartment

BTN3 YHR009C Btn3 Putative oxidoreductase involved in LE–Golgi transport; negative regulator of Btn2

Cytosol, LE

CLN1 ORF on human chromosome 1p32

PPT1 Palmitoyl protein thioesterase; lysosomal enzyme involved in infantile-onset NCL

Lysosome

CLN3 ORF on human chromosome 16p12

CLN3 Orthologue of yeast BTN1; involved in Batten disease (juvenile-onset NCL)

Golgi, endosomes, lysosomes

FUR4 YBR021W Fur4 Uracil permease PMGAS1 YMR307W Gas1 -1,3-glucanosyltransferase; required for cell wall assembly PM, cell wallGOS1 YHL031C Gos1 SNARE involved in intra-Golgi membrane transport GolgiHRR25 YPL204W Hrr25 Casein kinase involved in regulating vesicular trafficking,

DNA repair, and chromosome segregation; forms essential pair with Yck3

Golgi, PM

KAR2 YJL034W Kar2 ATPase involved in protein import into the ER; also acts as a chaperone to mediate protein folding

ER

KEX2 YNL238W Kex2 Subtilisin-like protease involved in the activation of proproteins of the secretory pathway

trans-Golgi

NYV1 YLR093C Nyv1 SNARE involved in vesicle fusion with vacuole Vacuolar membranePFA3 YNL326C Pfa3 Palmitoyltransferase for Vac8 required for vacuolar

membrane fusion; contains an Asp-His-His-Cys-cysteine rich (DHHC-CRD) domain

Vacuolar membrane

PRC1 YMR297W CPY1 Vacuolar CPY; sorted to vacuole via Vps10 Vacuolar lumenSEC1 YDR164C Sec1 SNARE regulator involved in docking and fusion of

exocytic vesiclesPM; sites of secretion

(bud neck and bud tip)SEC7 YDR170C Sec7 Guanine nucleotide exchange factor for Arf proteins;

involved in Golgi exporttrans-Golgi

SEC9 YGR009C Sec9 SNARE involved in fusion of secretory vesicles with PM PMSEC21 YNL287W Sec21 subunit of coatomer; involved in ER–Golgi transport ER, GolgiSEC22 YLR268W Sec22 SNARE involved in ER–Golgi transport ER, GolgiSED5 YLR026C Sed5 SNARE required for protein transport ER–Golgi,

intra-Golgi, and endosome–Golgi transportcis-Golgi

SFT1 YKL006C-A Sft1 SNARE required for intra-Golgi transport GolgiSNC1 YAL030W Snc1 SNARE involved in the fusion of secretory vesicles

with PM and endocytic vesicles with early endosomesPM, Golgi, endosomes

SNC2 YOR327C Snc2 SNARE involved in the fusion of secretory vesicles with PM and endocytic vesicles with early endosomes; paralog of Snc1

PM, Golgi, endosomes

SNX4 YJL036W Snx4 Sorting nexin involved in endosome–Golgi retrieval EndosomesSSO1 YPL232W Sso1 SNARE involved in the fusion of secretory vesicles with

PM and in prospore vesicle fusionPM

SSO2 YMR183C Sso2 SNARE involved in the fusion of secretory vesicles with PM; paralog of Sso1

PM

STE2 YFL026W Ste2 Receptor for -mating factor PMTLG1 YDR468C Tlg1 SNARE involved in the fusion of endocytic vesicles with

endosomes and endosome-derived vesicles with the late Golgi

Endosomes, trans-Golgi

TLG2 YOL018C Tlg2 SNARE involved in the fusion of endocytic vesicles with endosomes and endosome-derived vesicles with the late Golgi

Endosomes, trans-Golgi

VAC8 YEL013W Vac8 Palmitoylated vacuolar membrane protein required for the cytoplasm-to-vacuole targeting pathway

Vacuolar membrane

VAM3 YOR106W Vam3 SNARE involved in the fusion of membranes with the vacuole Vacuolar membrane

Btn1 regulates endosome–Golgi retrograde transport • Kama et al. S11

Table S9. Glossary of genes used in this study (Continued)

Gene name Systematic name Protein name Protein description Cellular localization

VPS5 YOR069W Vps5 Component of the retromer complex along with Vps35, Vps29, Vps17, and Vps26; essential for endosome–Golgi retrograde protein transport

LE, cytosol

VPS10 YBL017C Vps10 Type I transmembrane sorting receptor for multiple vacuolar hydrolases; cycles between the late Golgi and prevacuolar/endosomal compartments

Golgi, endosomes

VPS26 YJL053W Vps26 Component of the retromer complex along with Vps35, Vps29, Vps17, and Vps5; essential for endosome–Golgi retrograde transport

LE, cytosol

VPS27 YNR006W Vps27 Endosomal protein required for sorting ubiquitinated proteins destined for degradation in the vacuole

LE/MVB

VPS29 YHR012W Vps29 Component of the retromer complex along with Vps35, Vps26, Vps17, and Vps5; essential for endosome–Golgi retrograde protein transport

LE

VPS35 YJL154C Vps35 Component of the retromer complex along with Vps5, Vps29, Vps17, and Vps26; essential for endosome–Golgi retrograde protein transport

LE

VPS41 YDR080W Vps41 Subunit of the homotypic vacuole fusion and vacuole protein sorting complex; required for Golgi-to-endosome and endosome-to-vacuole transport

Endosome, vacuolar membrane

VTI1 YMR197C Vti1 SNARE involved in endosome–Golgi and endosome– vacuole transport

Endosomes, Golgi

YCK1 YHR135C Yck1 Palmitoylated casein kinase I involved in morphogenesis, septin assembly, endocytic trafficking; forms an essential pair with Yck2

PM, membranes

YCK2 YNL154C Yck2 Palmitoylated casein kinase I involved in morphogenesis, septin assembly, endocytic trafficking; forms an essential pair with Yck1

PM, membranes

YCK3 YER123W Yck3 Palmitoylated vacuolar membrane-localized casein kinase I; forms an essential pair with Hrr25; regulates AP-3 pathway

Vacuolar membrane

YIF1 YNL263C Yif1 Integral membrane protein involved in Rab GTPase regulation; required for ER–Golgi transport

Golgi

YKT6 YKL196C Ykt6 SNARE involved in trafficking ER–Golgi, intra-Golgi, and Golgi–vacuole transport

Golgi

YPT6 YLR262C Ypt6 Rab family GTPase involved in endosome–Golgi transport Golgi, endosomes

PM, plasma membrane.