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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 or deletion has opposing effects on Golgi morphology. (A) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5. WT, btn1Δ, and WT cells overexpressing BTN1 from a multicopy plasmid (BTN1) and all expressing GFP-Sed5 from a single-copy plasmid are shown. Note the large puncta in btn1Δ cells. (B) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5317A, whereas BTN1 overexpression enhances fragmentation (same as in A, except all strains express GFP-Sed5317A). Note the large puncta in WT and btn1Δ cells. (C) Golgi clustering is reduced in btn1Δ cells expressing GFP-Sed5317D (same as in A, except all strains express GFP-Sed5317D). (D) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5 from its genomic locus (GFP-Sed5 integrated [int.]). WT, btn1Δ, and WT cells overexpressing BTN1 from a multicopy plasmid (BTN1) and all expressing GFP-SED5 from its genomic locus are shown. Note the larger puncta in btn1Δ cells. (E) BTN1 overexpression reduces Golgi clustering in cells expressing GFP-Sed5317A from its genomic locus (same as in D, except all cells express GFP-SED5317A). Note the reduction in puncta size in cells overproducing Btn1. (F) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5317D from its genomic locus (same as in D, except all cells express GFP-SED5317D). Note the enhancement in puncta size in cells lacking BTN1. Bars, 1 µm.
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fig4: BTN1 overexpression or deletion has opposing effects on Golgi morphology. (A) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5. WT, btn1Δ, and WT cells overexpressing BTN1 from a multicopy plasmid (BTN1) and all expressing GFP-Sed5 from a single-copy plasmid are shown. Note the large puncta in btn1Δ cells. (B) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5317A, whereas BTN1 overexpression enhances fragmentation (same as in A, except all strains express GFP-Sed5317A). Note the large puncta in WT and btn1Δ cells. (C) Golgi clustering is reduced in btn1Δ cells expressing GFP-Sed5317D (same as in A, except all strains express GFP-Sed5317D). (D) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5 from its genomic locus (GFP-Sed5 integrated [int.]). WT, btn1Δ, and WT cells overexpressing BTN1 from a multicopy plasmid (BTN1) and all expressing GFP-SED5 from its genomic locus are shown. Note the larger puncta in btn1Δ cells. (E) BTN1 overexpression reduces Golgi clustering in cells expressing GFP-Sed5317A from its genomic locus (same as in D, except all cells express GFP-SED5317A). Note the reduction in puncta size in cells overproducing Btn1. (F) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5317D from its genomic locus (same as in D, except all cells express GFP-SED5317D). Note the enhancement in puncta size in cells lacking BTN1. Bars, 1 µm.

Mentions: First, we examined the effect of BTN1 overexpression or deletion in WT cells expressing GFP-tagged Sed5, Sed5317A, or Sed5317D from single-copy plasmids (Fig. 4; see Table S3 for the percentage of cells having Sed5 in dispersed/clustered Golgi). In cells expressing GFP-tagged Sed5, which yielded numerous variably sized puncta (Fig. 4 A, top row; Weinberger et al., 2005), we observed that the deletion of BTN1 led to an enhancement of their size (i.e., increased clustering; Fig. 4 A, bottom row). In cells expressing Sed5317A (Fig. 4 B, top row), which were previously shown to have greatly enlarged Golgi puncta (Weinberger et al., 2005), the deletion of BTN1 yielded even larger (although typically fewer) puncta (i.e., superclustering; Fig. 4 B, bottom row). Similar results were observed in btn1Δ cells expressing Sed5317D (Fig. 4 C, bottom row), although these puncta were smaller than in the other cells lacking BTN1 (Fig. 4, A and B [bottom rows]), probably as a result of the enhanced vesiculation exerted by Sed5317D (Weinberger et al., 2005). Conversely, BTN1 overexpression led to smaller and more dispersed puncta in all cell types (Fig. 4, A–C [middle rows]). Thus, Btn1 levels greatly modulate Golgi clustering. The notable changes in size and number of Golgi puncta likely result from an effect that Btn1 has upon native Sed5 that is present in the WT background (and expressed from the plasmid in Fig. 4 A).


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 or deletion has opposing effects on Golgi morphology. (A) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5. WT, btn1Δ, and WT cells overexpressing BTN1 from a multicopy plasmid (BTN1) and all expressing GFP-Sed5 from a single-copy plasmid are shown. Note the large puncta in btn1Δ cells. (B) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5317A, whereas BTN1 overexpression enhances fragmentation (same as in A, except all strains express GFP-Sed5317A). Note the large puncta in WT and btn1Δ cells. (C) Golgi clustering is reduced in btn1Δ cells expressing GFP-Sed5317D (same as in A, except all strains express GFP-Sed5317D). (D) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5 from its genomic locus (GFP-Sed5 integrated [int.]). WT, btn1Δ, and WT cells overexpressing BTN1 from a multicopy plasmid (BTN1) and all expressing GFP-SED5 from its genomic locus are shown. Note the larger puncta in btn1Δ cells. (E) BTN1 overexpression reduces Golgi clustering in cells expressing GFP-Sed5317A from its genomic locus (same as in D, except all cells express GFP-SED5317A). Note the reduction in puncta size in cells overproducing Btn1. (F) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5317D from its genomic locus (same as in D, except all cells express GFP-SED5317D). Note the enhancement in puncta size in cells lacking BTN1. Bars, 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: BTN1 overexpression or deletion has opposing effects on Golgi morphology. (A) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5. WT, btn1Δ, and WT cells overexpressing BTN1 from a multicopy plasmid (BTN1) and all expressing GFP-Sed5 from a single-copy plasmid are shown. Note the large puncta in btn1Δ cells. (B) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5317A, whereas BTN1 overexpression enhances fragmentation (same as in A, except all strains express GFP-Sed5317A). Note the large puncta in WT and btn1Δ cells. (C) Golgi clustering is reduced in btn1Δ cells expressing GFP-Sed5317D (same as in A, except all strains express GFP-Sed5317D). (D) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5 from its genomic locus (GFP-Sed5 integrated [int.]). WT, btn1Δ, and WT cells overexpressing BTN1 from a multicopy plasmid (BTN1) and all expressing GFP-SED5 from its genomic locus are shown. Note the larger puncta in btn1Δ cells. (E) BTN1 overexpression reduces Golgi clustering in cells expressing GFP-Sed5317A from its genomic locus (same as in D, except all cells express GFP-SED5317A). Note the reduction in puncta size in cells overproducing Btn1. (F) Deletion of BTN1 enhances Golgi clustering in cells expressing GFP-Sed5317D from its genomic locus (same as in D, except all cells express GFP-SED5317D). Note the enhancement in puncta size in cells lacking BTN1. Bars, 1 µm.
Mentions: First, we examined the effect of BTN1 overexpression or deletion in WT cells expressing GFP-tagged Sed5, Sed5317A, or Sed5317D from single-copy plasmids (Fig. 4; see Table S3 for the percentage of cells having Sed5 in dispersed/clustered Golgi). In cells expressing GFP-tagged Sed5, which yielded numerous variably sized puncta (Fig. 4 A, top row; Weinberger et al., 2005), we observed that the deletion of BTN1 led to an enhancement of their size (i.e., increased clustering; Fig. 4 A, bottom row). In cells expressing Sed5317A (Fig. 4 B, top row), which were previously shown to have greatly enlarged Golgi puncta (Weinberger et al., 2005), the deletion of BTN1 yielded even larger (although typically fewer) puncta (i.e., superclustering; Fig. 4 B, bottom row). Similar results were observed in btn1Δ cells expressing Sed5317D (Fig. 4 C, bottom row), although these puncta were smaller than in the other cells lacking BTN1 (Fig. 4, A and B [bottom rows]), probably as a result of the enhanced vesiculation exerted by Sed5317D (Weinberger et al., 2005). Conversely, BTN1 overexpression led to smaller and more dispersed puncta in all cell types (Fig. 4, A–C [middle rows]). Thus, Btn1 levels greatly modulate Golgi clustering. The notable changes in size and number of Golgi puncta likely result from an effect that Btn1 has upon native Sed5 that is present in the WT background (and expressed from the plasmid in Fig. 4 A).

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