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The yeast Batten disease orthologue Btn1 controls endosome-Golgi retrograde transport via SNARE assembly.

Kama R, Kanneganti V, Ungermann C, Gerst JE - J. Cell Biol. (2011)

Bottom Line: Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity.Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase.Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE-Golgi retrieval.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
The human Batten disease gene CLN3 and yeast orthologue BTN1 encode proteins of unclear function. We show that the loss of BTN1 phenocopies that of BTN2, which encodes a retromer accessory protein involved in the retrieval of specific cargo from late endosomes (LEs) to the Golgi. However, Btn1 localizes to Golgi and regulates soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) function to control retrograde transport. Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity. Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase. This may involve modification of the Yck3 lipid anchor, as substitution with a transmembrane domain suppresses the deletion of BTN1 and restores trafficking. Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE-Golgi retrieval. Thus, Btn1 controls retrograde sorting by regulating SNARE phosphorylation and assembly, a process that may be adversely affected in Batten Disease patients.

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BTN1 overexpression and deletion have opposing effects on Sed5 phosphorylation. (A) Sed5 is underphosphorylated in cells lacking BTN1 or YCK3. (left) WT cells, WT cells overexpressing BTN1 from a multicopy plasmid (BTN1), and btn1Δ cells were grown on glucose-containing medium. In parallel, cells expressing SED5 under a galactose-inducible promoter (GAL-SED5) were grown on galactose-containing medium and were shifted to glucose-containing medium for 16 h (to yield the sed5Δ condition). Cells were processed for Western analysis and 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 molecular mass/NP form of Sed5, whereas P indicates the higher molecular mass/P form. The histogram shows quantification (in arbitrary units) of the upper (P) and lower bands (NP) of Sed5 after normalization for Sso loading. (right) Cells expressing YCK3 under a GAL promoter (GAL-YCK3) were grown on galactose-containing medium and were shifted to glucose-containing medium for 8 h (to yield the yck3Δ condition) or were maintained on galactose-containing medium as a control. In parallel, cells expressing SED5 under a galactose-inducible promoter (GAL-SED5) were grown on galactose-containing medium and were shifted to glucose-containing medium for 16 h (to yield the sed5Δ condition). Cells were processed for Western analysis, and both Sed5 and Sso were detected as previously described. Note the presence of only the NP form of Sed5 (NP) in btn1Δ cells and in GAL-YCK3–expressing cells grown on glucose-containing medium. Also, note lack of Sed5 in GAL-SED5 cells grown on glucose-containing medium. The histogram shows quantification of the upper (P) and lower bands (NP) after normalization. The data shown are representative of multiple replicates of the experiment (n = 4). (B) Yif1 is mislocalized in the absence of Yck3. Yeast lacking YCK3 (yck3Δ) and expressing GFP-Yif1 from a single-copy plasmid were labeled with FM4-64. The top row depicts an endosomal pattern of labeling, whereas the bottom row depicts endosomal and vacuolar labeling. (C) Yif1 is localized to LEs in cells lacking YCK3. yck3Δ cells expressing GFP-Yif1 and RFP-Vps27 from single-copy plasmids are shown. (D) Kex2 is mislocalized in the absence of Yck3. yck3Δ yeast expressing Kex2-GFP from a single-copy plasmid and labeled with FM4-64 are shown. (E) The deletion of YCK3 enhances Golgi size. WT, btn1Δ, and yck3Δ cells expressing GFP-Sed5 and RFP-Vps27 from single-copy plasmids are shown. Note that the GFP-Sed5 and RFP-Vps27 signals do not overlap. (F) The double deletion of BTN1 and YCK3 does not further enhance Golgi size. btn1Δyck3Δ cells expressing GFP-Sed5 from a single-copy plasmid are shown. Bars, 1 µm.
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fig5: BTN1 overexpression and deletion have opposing effects on Sed5 phosphorylation. (A) Sed5 is underphosphorylated in cells lacking BTN1 or YCK3. (left) WT cells, WT cells overexpressing BTN1 from a multicopy plasmid (BTN1), and btn1Δ cells were grown on glucose-containing medium. In parallel, cells expressing SED5 under a galactose-inducible promoter (GAL-SED5) were grown on galactose-containing medium and were shifted to glucose-containing medium for 16 h (to yield the sed5Δ condition). Cells were processed for Western analysis and 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 molecular mass/NP form of Sed5, whereas P indicates the higher molecular mass/P form. The histogram shows quantification (in arbitrary units) of the upper (P) and lower bands (NP) of Sed5 after normalization for Sso loading. (right) Cells expressing YCK3 under a GAL promoter (GAL-YCK3) were grown on galactose-containing medium and were shifted to glucose-containing medium for 8 h (to yield the yck3Δ condition) or were maintained on galactose-containing medium as a control. In parallel, cells expressing SED5 under a galactose-inducible promoter (GAL-SED5) were grown on galactose-containing medium and were shifted to glucose-containing medium for 16 h (to yield the sed5Δ condition). Cells were processed for Western analysis, and both Sed5 and Sso were detected as previously described. Note the presence of only the NP form of Sed5 (NP) in btn1Δ cells and in GAL-YCK3–expressing cells grown on glucose-containing medium. Also, note lack of Sed5 in GAL-SED5 cells grown on glucose-containing medium. The histogram shows quantification of the upper (P) and lower bands (NP) after normalization. The data shown are representative of multiple replicates of the experiment (n = 4). (B) Yif1 is mislocalized in the absence of Yck3. Yeast lacking YCK3 (yck3Δ) and expressing GFP-Yif1 from a single-copy plasmid were labeled with FM4-64. The top row depicts an endosomal pattern of labeling, whereas the bottom row depicts endosomal and vacuolar labeling. (C) Yif1 is localized to LEs in cells lacking YCK3. yck3Δ cells expressing GFP-Yif1 and RFP-Vps27 from single-copy plasmids are shown. (D) Kex2 is mislocalized in the absence of Yck3. yck3Δ yeast expressing Kex2-GFP from a single-copy plasmid and labeled with FM4-64 are shown. (E) The deletion of YCK3 enhances Golgi size. WT, btn1Δ, and yck3Δ cells expressing GFP-Sed5 and RFP-Vps27 from single-copy plasmids are shown. Note that the GFP-Sed5 and RFP-Vps27 signals do not overlap. (F) The double deletion of BTN1 and YCK3 does not further enhance Golgi size. btn1Δyck3Δ cells expressing GFP-Sed5 from a single-copy plasmid are shown. Bars, 1 µm.

Mentions: Because the deletion of BTN1 enhances Golgi size (Fig. 4), which corresponds to the NP form of Sed5 (Weinberger et al., 2005), and BTN1 overexpression has an opposite effect that corresponds with phosphorylated (P) Sed5 (Weinberger et al., 2005), we examined Sed5 phosphorylation in cells lacking or overexpressing BTN1 by Western blotting with anti-Sed5 antibodies. Cell extracts from WT, BTN1-overexpressing, and btn1Δ cells were resolved on 11.5% acrylamide gels and were probed with anti-Sed5 antibodies to reveal the lower (NP) and higher (P) molecular mass forms (Weinberger et al., 2005). As previously seen, Sed5 can exist in the P form in WT cells (Weinberger et al., 2005), and this signal appeared somewhat stronger (≥15%) in WT cells overexpressing BTN1 than in WT cells (see representative experiment shown in Fig. 5 A, top and bottom left). More strikingly, the P form of Sed5 was greatly reduced in btn1Δ cells, and quantification revealed that its levels were three- to fourfold lower than in WT cells after normalization for loading (Fig. 5 A, bottom left), although a small reduction in the levels of Sed5 protein was also apparent. The NP/P ratio for Sed5 in WT cells, WT cells overexpressing BTN1, and btn1Δ cells was 2.7:1, 2.2:1, and 5:1 in this representative experiment (NP/P ratio for btn1Δ cells was 7.2 ± 1.7:1 in four experiments; see Figs. 6 (C and F) and S5 A for similar results). Thus, excess Btn1 appears to enhance Sed5 phosphorylation, whereas its absence greatly enhances the NP state. These results could account for the changes in the aforementioned Golgi morphology observed (Fig. 4).


The yeast Batten disease orthologue Btn1 controls endosome-Golgi retrograde transport via SNARE assembly.

Kama R, Kanneganti V, Ungermann C, Gerst JE - J. Cell Biol. (2011)

BTN1 overexpression and deletion have opposing effects on Sed5 phosphorylation. (A) Sed5 is underphosphorylated in cells lacking BTN1 or YCK3. (left) WT cells, WT cells overexpressing BTN1 from a multicopy plasmid (BTN1), and btn1Δ cells were grown on glucose-containing medium. In parallel, cells expressing SED5 under a galactose-inducible promoter (GAL-SED5) were grown on galactose-containing medium and were shifted to glucose-containing medium for 16 h (to yield the sed5Δ condition). Cells were processed for Western analysis and 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 molecular mass/NP form of Sed5, whereas P indicates the higher molecular mass/P form. The histogram shows quantification (in arbitrary units) of the upper (P) and lower bands (NP) of Sed5 after normalization for Sso loading. (right) Cells expressing YCK3 under a GAL promoter (GAL-YCK3) were grown on galactose-containing medium and were shifted to glucose-containing medium for 8 h (to yield the yck3Δ condition) or were maintained on galactose-containing medium as a control. In parallel, cells expressing SED5 under a galactose-inducible promoter (GAL-SED5) were grown on galactose-containing medium and were shifted to glucose-containing medium for 16 h (to yield the sed5Δ condition). Cells were processed for Western analysis, and both Sed5 and Sso were detected as previously described. Note the presence of only the NP form of Sed5 (NP) in btn1Δ cells and in GAL-YCK3–expressing cells grown on glucose-containing medium. Also, note lack of Sed5 in GAL-SED5 cells grown on glucose-containing medium. The histogram shows quantification of the upper (P) and lower bands (NP) after normalization. The data shown are representative of multiple replicates of the experiment (n = 4). (B) Yif1 is mislocalized in the absence of Yck3. Yeast lacking YCK3 (yck3Δ) and expressing GFP-Yif1 from a single-copy plasmid were labeled with FM4-64. The top row depicts an endosomal pattern of labeling, whereas the bottom row depicts endosomal and vacuolar labeling. (C) Yif1 is localized to LEs in cells lacking YCK3. yck3Δ cells expressing GFP-Yif1 and RFP-Vps27 from single-copy plasmids are shown. (D) Kex2 is mislocalized in the absence of Yck3. yck3Δ yeast expressing Kex2-GFP from a single-copy plasmid and labeled with FM4-64 are shown. (E) The deletion of YCK3 enhances Golgi size. WT, btn1Δ, and yck3Δ cells expressing GFP-Sed5 and RFP-Vps27 from single-copy plasmids are shown. Note that the GFP-Sed5 and RFP-Vps27 signals do not overlap. (F) The double deletion of BTN1 and YCK3 does not further enhance Golgi size. btn1Δyck3Δ cells expressing GFP-Sed5 from a single-copy plasmid are shown. Bars, 1 µm.
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fig5: BTN1 overexpression and deletion have opposing effects on Sed5 phosphorylation. (A) Sed5 is underphosphorylated in cells lacking BTN1 or YCK3. (left) WT cells, WT cells overexpressing BTN1 from a multicopy plasmid (BTN1), and btn1Δ cells were grown on glucose-containing medium. In parallel, cells expressing SED5 under a galactose-inducible promoter (GAL-SED5) were grown on galactose-containing medium and were shifted to glucose-containing medium for 16 h (to yield the sed5Δ condition). Cells were processed for Western analysis and 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 molecular mass/NP form of Sed5, whereas P indicates the higher molecular mass/P form. The histogram shows quantification (in arbitrary units) of the upper (P) and lower bands (NP) of Sed5 after normalization for Sso loading. (right) Cells expressing YCK3 under a GAL promoter (GAL-YCK3) were grown on galactose-containing medium and were shifted to glucose-containing medium for 8 h (to yield the yck3Δ condition) or were maintained on galactose-containing medium as a control. In parallel, cells expressing SED5 under a galactose-inducible promoter (GAL-SED5) were grown on galactose-containing medium and were shifted to glucose-containing medium for 16 h (to yield the sed5Δ condition). Cells were processed for Western analysis, and both Sed5 and Sso were detected as previously described. Note the presence of only the NP form of Sed5 (NP) in btn1Δ cells and in GAL-YCK3–expressing cells grown on glucose-containing medium. Also, note lack of Sed5 in GAL-SED5 cells grown on glucose-containing medium. The histogram shows quantification of the upper (P) and lower bands (NP) after normalization. The data shown are representative of multiple replicates of the experiment (n = 4). (B) Yif1 is mislocalized in the absence of Yck3. Yeast lacking YCK3 (yck3Δ) and expressing GFP-Yif1 from a single-copy plasmid were labeled with FM4-64. The top row depicts an endosomal pattern of labeling, whereas the bottom row depicts endosomal and vacuolar labeling. (C) Yif1 is localized to LEs in cells lacking YCK3. yck3Δ cells expressing GFP-Yif1 and RFP-Vps27 from single-copy plasmids are shown. (D) Kex2 is mislocalized in the absence of Yck3. yck3Δ yeast expressing Kex2-GFP from a single-copy plasmid and labeled with FM4-64 are shown. (E) The deletion of YCK3 enhances Golgi size. WT, btn1Δ, and yck3Δ cells expressing GFP-Sed5 and RFP-Vps27 from single-copy plasmids are shown. Note that the GFP-Sed5 and RFP-Vps27 signals do not overlap. (F) The double deletion of BTN1 and YCK3 does not further enhance Golgi size. btn1Δyck3Δ cells expressing GFP-Sed5 from a single-copy plasmid are shown. Bars, 1 µm.
Mentions: Because the deletion of BTN1 enhances Golgi size (Fig. 4), which corresponds to the NP form of Sed5 (Weinberger et al., 2005), and BTN1 overexpression has an opposite effect that corresponds with phosphorylated (P) Sed5 (Weinberger et al., 2005), we examined Sed5 phosphorylation in cells lacking or overexpressing BTN1 by Western blotting with anti-Sed5 antibodies. Cell extracts from WT, BTN1-overexpressing, and btn1Δ cells were resolved on 11.5% acrylamide gels and were probed with anti-Sed5 antibodies to reveal the lower (NP) and higher (P) molecular mass forms (Weinberger et al., 2005). As previously seen, Sed5 can exist in the P form in WT cells (Weinberger et al., 2005), and this signal appeared somewhat stronger (≥15%) in WT cells overexpressing BTN1 than in WT cells (see representative experiment shown in Fig. 5 A, top and bottom left). More strikingly, the P form of Sed5 was greatly reduced in btn1Δ cells, and quantification revealed that its levels were three- to fourfold lower than in WT cells after normalization for loading (Fig. 5 A, bottom left), although a small reduction in the levels of Sed5 protein was also apparent. The NP/P ratio for Sed5 in WT cells, WT cells overexpressing BTN1, and btn1Δ cells was 2.7:1, 2.2:1, and 5:1 in this representative experiment (NP/P ratio for btn1Δ cells was 7.2 ± 1.7:1 in four experiments; see Figs. 6 (C and F) and S5 A for similar results). Thus, excess Btn1 appears to enhance Sed5 phosphorylation, whereas its absence greatly enhances the NP state. These results could account for the changes in the aforementioned Golgi morphology observed (Fig. 4).

Bottom Line: Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity.Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase.Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE-Golgi retrieval.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
The human Batten disease gene CLN3 and yeast orthologue BTN1 encode proteins of unclear function. We show that the loss of BTN1 phenocopies that of BTN2, which encodes a retromer accessory protein involved in the retrieval of specific cargo from late endosomes (LEs) to the Golgi. However, Btn1 localizes to Golgi and regulates soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) function to control retrograde transport. Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity. Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase. This may involve modification of the Yck3 lipid anchor, as substitution with a transmembrane domain suppresses the deletion of BTN1 and restores trafficking. Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE-Golgi retrieval. Thus, Btn1 controls retrograde sorting by regulating SNARE phosphorylation and assembly, a process that may be adversely affected in Batten Disease patients.

Show MeSH
Related in: MedlinePlus