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First published online 19 March 2009
doi: 10.1242/jcs.038323

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S. pombe btn1, the orthologue of the Batten disease gene CLN3, is required for vacuole protein sorting of Cpy1p and Golgi exit of Vps10p

¹ MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK
² Molecular Medicine Unit, UCL Institute of Child Health, University College London, London WC1E 6BT, UK
³ Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK

View larger version (18K): [in this window] [in a new window]   Fig. 1. btn1 is required for vacuole protein sorting of the vacuole peptidase Cpy1p. Colony blot assay of indicated cells grown for 6 hours on YES agar using S. pombe specific anti-Cpy1p antibody. btn1 cells abnormally secrete Cpy1p. (A) Colony blot of indicated cells showing minimal Cpy1 secretion from wild-type (wt) cells but secretion from btn1, vps26 and vps10 cells. (B) Bar chart depicting mean (± s.d.) density values relative to wild-type levels of Cpy1p secretion in colony blot assays (mean of three experiments).

View larger version (36K): [in this window] [in a new window]   Fig. 2. btn1 mediates exit of Vps10p from the Golgi. (A,B) Delayed trafficking of Vps10p in btn1 cells: fluorescence images of wild-type (wt, A) or btn1 cells (B) expressing Vps10p-YFP showing defective trafficking in btn1 cells (mainly ER or Golgi rather than the TGN distribution typical of wild-type cells). (C,D) Vps10p is prevented from entering the TGN-to-vacuole pathway in the absence of btn1: fluorescence images of wild-type (wt, C) or btn1 cells (D) overexpressing Vps10p-YFP after 3 hours of thiamine-induced promoter repression and FM4-64 vacuole labelling showing Vps10p is retained within cytoplasmic Golgi-like compartments in btn1 cells. (E,F,G) Vps10p is delayed within the Golgi in the absence of btn1. Fluorescence images of btn1 cells expressing Vps10p-YFP after 3 hours of thiamine-induced promoter repression (E) and after 1 hour of Brefeldin A (BFA) treatment (F) showing that these Vps10p-containing compartments are not post-Golgi because they collapse back to the ER. Vps10p can exit the Golgi in wild-type cells. (G) Fluorescence images of wild-type (wt) cells expressing Vps10p-YFP after 3 hours of thiamine-induced promoter repression and 1 hour of Brefeldin A (BFA) treatment, showing that these Vps10p-containing compartments are not Golgi because they are unaffected by BFA treatment. Scale bars: 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 3. Btn1 localises to the Golgi compartment. (A) GFP-Btn1p colocalises extensively with Gms1p-CFP. Confocal images showing wild-type cells expressing Gms1p-CFP and GFP-Btn1p (asterisk indicates a cell expressing GFP-Btn1p at low levels, and the arrowhead a puncta magnified three times in the inset). (B) Bar chart depicting percentage of compartments (n>200) containing indicated proteins. (C) GFP-Btn1pG136A mutant protein does not colocalise with Gms1p. Confocal images of wild-type cells expressing Gms1p-CFP and GFP-Btn1pG136A. Scale bars: 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 4. Btn1p localises upstream of Vps10p. (A,B) Btn1p localises to the Golgi compartment in wild-type cells, which is upstream of Vps10p. Fluorescence images of wild-type cells expressing GFP-Btn1p (A) or Vps10p-YFP (B) treated with Brefeldin A (BFA) or without (DMSO control) showing that Btn1p-containing Golgi compartments collapse back to the ER whereas Vps10p-containing compartments do not. (C) Most Vps10p is not located at the Golgi. Confocal images showing wild-type cells expressing Gms1p-CFP and Vps10p-YFP that do not colocalise. (D) Bar chart depicting percentage of compartments (n>200) containing indicated proteins. (E,F) Unlike Vps10p, Btn1p remains Golgi localised in the absence of a functional retromer complex, and is not misrouted to the vacuole. GFP-Btn1p (E) or Vps10p-YFP (F) expressed in vps26 cells and treated with BFA or without (DMSO control). All Btn1p-containing Golgi compartments collapse back to the ER whereas Vps10p-containing vacuole compartments do not. Scale bars: 10 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Btn1p regulates Golgi number and positioning. Absence of btn1 causes a reduction in the number of Gms1p-stained Golgi compartments. (A) Confocal images of wild-type (wt) btn1 cells expressing Gms1p-CFP showing reduced numbers of Golgi and positioning defects (end panel shows a single typical cell). (B) Bar chart of numbers of Golgi in wild-type (wt), btn1 cells and wild-type cells overexpressing Btn1p (n>200 Golgi across single cross-sections in three independent experiments); wild-type cells, 7.1±2.2; btn1 cells, 4.2±1.4; cells overexpressing Btn1p, 5.9±2.59; ***P<0.001. (C) Absence of btn1 or ectopic expression of Btn1p alters the distribution of Golgi compartments. Bar chart of positions of Gms1p-stained Golgi compartments in wild-type (wt), btn1 cells and cells overexpressing Btn1p with respect to their distance from the nucleus. The cartoon in A illustrates the positions of regions counted (n>200 Golgi over three experiments). Scale bars: 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 6. btn1 affects Golgi morphology. (A,B) Typical Golgi in wild-type (wt) cells have electron-dense ribbon-like flattened cisternae, often arranged in stacks. (A) Electron micrograph of a single cell at log phase, showing distribution of multiple Golgi (G, Golgi; L, lipid droplet; M, mitochondrion; N, nucleus; V, vacuole). Scale bar: 2 µm. (B) Electron micrographs of Golgi with single or multiple adjacent cisternae. Scale bar: 0.5 µm. (C,D) Typical Golgi in btn1 cells are longer and have fewer stacks, or have swollen and mis-shapen cisternae. (C) Electron micrographs of single cells at log phase showing fewer and less-prominent Golgi. Scale bars: 2 µm. (D) Electron micrographs of recognisable Golgi with stacked cisternae, and examples of larger aberrant Golgi structures. Scale bar: 0.5 µm. (E) btn1 cells have many atypical Golgi and fewer Golgi with multiple stacked cisternae. Bar chart of % frequency of Golgi complexes with defined numbers of stacks (1, 2, 3 or more), or those with aberrant morphology, in log-phase wild-type and btn1 cells [wt, n=43 Golgi (21 cells); btn1, n=48 Golgi (28 cells)].

View larger version (35K): [in this window] [in a new window]   Fig. 7. btn1 might be required for other vps pathways. (A) Btn1 might also be involved in a second vacuole protein-sorting pathway for Cpy1p. Colony blot assay of indicated cells grown on YES agar plates showing increased Cpy1p secretion from a vps10btn1 double deletion strain compared to vps10. (B) Colony blot assay of indicated double deletion strains showing that vps26 and vps26btn1 cells secrete similar amounts to parental strains, but that vps34btn1 cells secrete more than parental strains as observed in C at a lower exposure. (D) Bar chart depicting mean density values relative to wild-type levels of Cpy1p secretion in colony blot assays (mean ± s.d. of three experiments). *P<0.05.

View larger version (28K): [in this window] [in a new window]   Fig. 8. Btn1p suppresses the Cpy1p secretion defect of vps34 cells. Ectopic expression of Btn1p rescues the Cpy1p secretion defect of vps34 cells but not that of vps26 or vps10 cells. (A) Colony blot assay of indicated cells grown on MM agar plates plus supplements for 6 hours. (B) Bar chart depicting mean density values relative to wild-type level of Cpy1p secretion of colony blot assays from indicated cells (mean of two experiments). (C-F) The rescue of the Cpy1p secretion defect of vps34 cells is specific to native Btn1p protein and to levels of protein expression: colony blot assay of indicated cells grown on MM agar plates plus supplements for 6 hours with native (Btn1p) or mutant Btn1pG136A protein in the absence (C) or presence (D) of thiamine (to repress expression). Bar charts depicting mean density values relative to wild-type level of Cpy1p secretion of colony blot assays from indicated cells without (E) and with (F) thiamine-induced promoter repression (mean ± s.d. of four and two experiments, respectively). **P<0.01.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Diagrammatic summary of the effect of loss of btn1 on Vps10p trafficking and Cpy1p sorting. When btn1 is deleted (right panel), Vps10p trafficking beyond the Golgi compartment to the TGN is impeded. Consequently, a proportion of Cpy1p is mis-sorted at the TGN and secreted. Black arrows represent established trafficking pathways. Green arrows in left panel represent the Vps10p trafficking or recycling pathway. BFA treatment causes compartments to the left of the dotted line to collapse back to the ER. Sec, secretory vesicles; Prevac, prevacuole compartment that is most likely a late endosome or multivesicular body; Vac, vacuole.

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