JCS038323 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Figure S1 -

  Fig. S1. The ER in S. pombe is perinuclear and cortical, with connective tubules. Fluorescence image of wild-type cells (wt) endogenously expressing ER marker Ost1p-GFP, showing a typical ER pattern of localisation: perinuclear and cortica,l with ribbon-like connective tubules. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. The effect of BFA treatment on Golgi-resident proteins in S. pombe. Brefeldin A (BFA) treatment causes Golgi compartments to collapse back to the ER. (A) Confocal image of wild-type (wt) cells ectopically expressing Gms1p-CFP and treated with BFA, showing Gms1p-containing Golgi compartments collapse back to the ER. (B) Maximum projection confocal image of wild-type (wt) cells endogenously expressing Anp1p-GFP (i), and confocal image of those cells treated with BFA, showing Anp1p-containing Golgi compartments collapse back to the ER (ii). Scale bar: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Btn1p does not recycle via the retromer complex. Vps10p requires retromer to recycle back to the TGN. (A) Fluorescence image of vps26Δ cells expressing Vps10p-YFP showing that Vps10p-YFP is mislocalised to the vacuoles in vps26Δ cells (i); (ii) fluorescence images of vps26Δ cells expressing Vps10p-YFP and with FM4-64 vacuole labelling (30 minutes plus chase), confirming vacuole localisation. (B) GFP-Btn1p does not recycle using retromer. Fluorescence image of vps26Δ cells expressing GFP-Btn1p showing GFP-Btn1p is localised to cytoplasmic punctae and is not mislocalised to the vacuoles in vps26Δ cells. (C) Overexpressed GFP-Btn1p does eventually traffic to the vacuole in wild-type cells. Fluorescence image showing wild-type (wt) cells expressing GFP-Btn1p after 6 hours of thiamine-induced promoter repression and with FM4-64 vacuole labelling. (D) Post-Golgi vacuole location of Btn1p is insensitive to BFA treatment. Fluorescence image of wild-type cells expressing GFP-Btn1p after 3 hours of thiamine-induced promoter repression and after BFA treatment. Scale bars: 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Btn1p regulates Golgi number and positioning. Absence of btn1 causes a reduction in the total number and altered distribution of Gms1p-stained Golgi compartments. Maximum projection images of confocal z-stacks of wild-type (wt) or btn1Δ cells expressing Gms1p-CFP. Arrowheads indicate Golgi compartments adjacent to the nucleus in wild-type cells that are mainly absent in btn1Δ cells. Scale bars: 10 µm.

• Supplemental Figure S5 -

  Fig. S5. Btn1 localises to the Golgi compartment in btn1Δ cells and vps34Δ cells. (A) Ectopic expression of Btn1p rescues the Cpy1 secretion defect of btn1Δ cells. Colony blot assay of indicated cells grown on MM agar plates plus supplements for 6 hours; expression from the parental GFP pREP41 vector is the negative control (−). Ectopic expression of CLN3 rescues the Cpy1 secretion defect of btn1Δ cells: colony blot assay of indicated cells grown on MM agar plates plus supplements for 6 hours, expression from the parental GFP pREP42 vector is the negative control (−). (B) GFP-Btn1p localises to BFA-sensitive Golgi in btn1Δ cells. Fluorescence image of btn1Δ cells expressing GFP-Btn1p and treated with BFA showing compartments collapse back to the ER, as in wild-type cells. (C) GFP-Btn1p localises to the Golgi in vps34Δ cells. Fluorescence images of vps34Δ cells expressing GFP-Btn1p (i), and treated with BFA (ii), showing that most compartments collapse back to the ER. (D) Mutant Btn1p^G136A protein localises to the ER in vps34Δ cells. Fluorescence image of vps34Δ cells expressing mutant Btn1p^G136A protein in typical ER-like compartments that are not BFA sensitive (not shown). Scale bars: 10 µm.

• Supplemental Figure S6 -

  Fig. S6. Btn1 expression verification in cells used in colony blot assays. (A) Ectopic expression of either GFP or GFP-Btn1p in wild-type, vps10Δ, vps34Δ and vps26Δ cells. (B) Ectopic expression of GFP, GFP-Btn1p or GFP-Btn1p^G136A in vps34Δ cells with and without thiamine (4 µM to repress expression). Scale bars: 10 µm.

• Movie 1 -

  Movie 1. Btn1p regulates Golgi number and positioning. Z-stacks of wild-type cells expressing Gms1p-YFP (same cells as in supplementary material Fig. S4).

• Movie 2 -

  Movie 2. Btn1p regulates Golgi number and positioning. Z-stacks of btn1Δ cells expressing Gms1p-YFP (same cells as in supplementary material Fig. S4).