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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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Yck3 palmitoylation is required for LE–Golgi sorting but can be obviated upon substitution with a TMD. (A) Palmitoyltransferase activity is required for LE–Golgi retrieval. Yeast lacking five DHHC palmitoyltransferases (DHHC 5×Δ) and expressing either GFP-YIF1 (top) or KEX2-GFP (bottom) are shown. Note the localization of Yif1 to LE and vacuole and Kex2 to the vacuole membrane. (B) Yck3 is mislocalized to LEs in cells depleted of palmitoyltransferases. DHHC 5×Δ yeast expressing GFP-YCK3 are shown. (C) Depletion of DHHC palmitoyl transferases reduces Sed5 phosphorylation. WT, btn1Δ, and DHHC 5×Δ cells were processed for Western analysis with anti-Sed5 and -Sso antibodies, as described in Fig. 5 A. NP and P indicate the NP and P form of Sed5, respectively. The histogram shows quantification of the Sed5 bands after normalization. The data shown are representative of multiple replicates of the experiment (n = 3). (D) Yif1 is mislocalized to LEs in yck3Δ cells expressing a Yck3-FYVE fusion protein. WT and btn1Δ cells expressing both YCK3-FYVE from the YCK3 locus and GFP-YIF1 from a single-copy plasmid are shown. (E) Expression of Yck3 fused to a TMD bypasses the Btn1 requirement for Yif1 localization to the Golgi. btn1Δ cells and btn1Δ cells expressing YCK3-VAM3 from the YCK3 locus were transformed with a plasmid expressing GFP-Yif1, labeled with FM4-64, and examined. Note the localization of Yif1 to LEs in btn1Δ cells and to smaller puncta (that do not colabel with FM4-64) in cells expressing Yck3-Vam3. (F) Expression of Yck3-Vam3 partially restores Sed phosphorylation. WT, btn1Δ, and btn1Δ cells expressing YCK3-VAM3 were processed for Western analysis and Sed5 quantification, as described in Fig. 5 A. The data shown are representative of multiple replicates of the experiment (n = 2). Bars, 1 µm.
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fig6: Yck3 palmitoylation is required for LE–Golgi sorting but can be obviated upon substitution with a TMD. (A) Palmitoyltransferase activity is required for LE–Golgi retrieval. Yeast lacking five DHHC palmitoyltransferases (DHHC 5×Δ) and expressing either GFP-YIF1 (top) or KEX2-GFP (bottom) are shown. Note the localization of Yif1 to LE and vacuole and Kex2 to the vacuole membrane. (B) Yck3 is mislocalized to LEs in cells depleted of palmitoyltransferases. DHHC 5×Δ yeast expressing GFP-YCK3 are shown. (C) Depletion of DHHC palmitoyl transferases reduces Sed5 phosphorylation. WT, btn1Δ, and DHHC 5×Δ cells were processed for Western analysis with anti-Sed5 and -Sso antibodies, as described in Fig. 5 A. NP and P indicate the NP and P form of Sed5, respectively. The histogram shows quantification of the Sed5 bands after normalization. The data shown are representative of multiple replicates of the experiment (n = 3). (D) Yif1 is mislocalized to LEs in yck3Δ cells expressing a Yck3-FYVE fusion protein. WT and btn1Δ cells expressing both YCK3-FYVE from the YCK3 locus and GFP-YIF1 from a single-copy plasmid are shown. (E) Expression of Yck3 fused to a TMD bypasses the Btn1 requirement for Yif1 localization to the Golgi. btn1Δ cells and btn1Δ cells expressing YCK3-VAM3 from the YCK3 locus were transformed with a plasmid expressing GFP-Yif1, labeled with FM4-64, and examined. Note the localization of Yif1 to LEs in btn1Δ cells and to smaller puncta (that do not colabel with FM4-64) in cells expressing Yck3-Vam3. (F) Expression of Yck3-Vam3 partially restores Sed phosphorylation. WT, btn1Δ, and btn1Δ cells expressing YCK3-VAM3 were processed for Western analysis and Sed5 quantification, as described in Fig. 5 A. The data shown are representative of multiple replicates of the experiment (n = 2). 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)

Yck3 palmitoylation is required for LE–Golgi sorting but can be obviated upon substitution with a TMD. (A) Palmitoyltransferase activity is required for LE–Golgi retrieval. Yeast lacking five DHHC palmitoyltransferases (DHHC 5×Δ) and expressing either GFP-YIF1 (top) or KEX2-GFP (bottom) are shown. Note the localization of Yif1 to LE and vacuole and Kex2 to the vacuole membrane. (B) Yck3 is mislocalized to LEs in cells depleted of palmitoyltransferases. DHHC 5×Δ yeast expressing GFP-YCK3 are shown. (C) Depletion of DHHC palmitoyl transferases reduces Sed5 phosphorylation. WT, btn1Δ, and DHHC 5×Δ cells were processed for Western analysis with anti-Sed5 and -Sso antibodies, as described in Fig. 5 A. NP and P indicate the NP and P form of Sed5, respectively. The histogram shows quantification of the Sed5 bands after normalization. The data shown are representative of multiple replicates of the experiment (n = 3). (D) Yif1 is mislocalized to LEs in yck3Δ cells expressing a Yck3-FYVE fusion protein. WT and btn1Δ cells expressing both YCK3-FYVE from the YCK3 locus and GFP-YIF1 from a single-copy plasmid are shown. (E) Expression of Yck3 fused to a TMD bypasses the Btn1 requirement for Yif1 localization to the Golgi. btn1Δ cells and btn1Δ cells expressing YCK3-VAM3 from the YCK3 locus were transformed with a plasmid expressing GFP-Yif1, labeled with FM4-64, and examined. Note the localization of Yif1 to LEs in btn1Δ cells and to smaller puncta (that do not colabel with FM4-64) in cells expressing Yck3-Vam3. (F) Expression of Yck3-Vam3 partially restores Sed phosphorylation. WT, btn1Δ, and btn1Δ cells expressing YCK3-VAM3 were processed for Western analysis and Sed5 quantification, as described in Fig. 5 A. The data shown are representative of multiple replicates of the experiment (n = 2). Bars, 1 µm.
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fig6: Yck3 palmitoylation is required for LE–Golgi sorting but can be obviated upon substitution with a TMD. (A) Palmitoyltransferase activity is required for LE–Golgi retrieval. Yeast lacking five DHHC palmitoyltransferases (DHHC 5×Δ) and expressing either GFP-YIF1 (top) or KEX2-GFP (bottom) are shown. Note the localization of Yif1 to LE and vacuole and Kex2 to the vacuole membrane. (B) Yck3 is mislocalized to LEs in cells depleted of palmitoyltransferases. DHHC 5×Δ yeast expressing GFP-YCK3 are shown. (C) Depletion of DHHC palmitoyl transferases reduces Sed5 phosphorylation. WT, btn1Δ, and DHHC 5×Δ cells were processed for Western analysis with anti-Sed5 and -Sso antibodies, as described in Fig. 5 A. NP and P indicate the NP and P form of Sed5, respectively. The histogram shows quantification of the Sed5 bands after normalization. The data shown are representative of multiple replicates of the experiment (n = 3). (D) Yif1 is mislocalized to LEs in yck3Δ cells expressing a Yck3-FYVE fusion protein. WT and btn1Δ cells expressing both YCK3-FYVE from the YCK3 locus and GFP-YIF1 from a single-copy plasmid are shown. (E) Expression of Yck3 fused to a TMD bypasses the Btn1 requirement for Yif1 localization to the Golgi. btn1Δ cells and btn1Δ cells expressing YCK3-VAM3 from the YCK3 locus were transformed with a plasmid expressing GFP-Yif1, labeled with FM4-64, and examined. Note the localization of Yif1 to LEs in btn1Δ cells and to smaller puncta (that do not colabel with FM4-64) in cells expressing Yck3-Vam3. (F) Expression of Yck3-Vam3 partially restores Sed phosphorylation. WT, btn1Δ, and btn1Δ cells expressing YCK3-VAM3 were processed for Western analysis and Sed5 quantification, as described in Fig. 5 A. The data shown are representative of multiple replicates of the experiment (n = 2). 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