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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 localizes to the Golgi in a manner dependent on Btn2. (A) Btn1 does not colocalize with FM4-64–labeled compartments. (top) WT cells expressing GFP-tagged BTN1 from its genomic locus under the control of a GAL promoter (GAL integrated [GAL int]) were grown on galactose-containing medium and were labeled with FM4-64. (middle) WT cells expressing GFP-BTN1 constitutively from a single-copy plasmid (CEN plasmid) were grown on glucose-containing medium and were labeled with FM4-64. (bottom) WT cells expressing GFP-BTN1 from its genomic locus and under the control of the BTN1 promoter were grown on glucose-containing medium and were labeled with FM4-64. (B) Btn1 colocalizes with Yif1. WT cells expressing GFP-BTN1 from its genomic locus under the control of a GAL promoter and either RFP-YIF1 from a single-copy plasmid (top row) or SEC7-DsRed from a chromosomal integration (bottom row) were grown on galactose-containing medium and visualized. (C) Btn1 and Btn2 do not colocalize. WT cells expressing GFP-BTN1 from its chromosomal locus under the control of a GAL promoter and BTN2-RFP from a single-copy plasmid are shown; cells were grown on galactose-containing medium. (D) Btn1 is mislocalized to the vacuole in btn2Δ cells. btn2Δ cells expressing GFP-BTN1 from a single-copy plasmid were grown on glucose-containing medium and were labeled with FM4-64. Bars, 1 µm.
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fig2: Btn1 localizes to the Golgi in a manner dependent on Btn2. (A) Btn1 does not colocalize with FM4-64–labeled compartments. (top) WT cells expressing GFP-tagged BTN1 from its genomic locus under the control of a GAL promoter (GAL integrated [GAL int]) were grown on galactose-containing medium and were labeled with FM4-64. (middle) WT cells expressing GFP-BTN1 constitutively from a single-copy plasmid (CEN plasmid) were grown on glucose-containing medium and were labeled with FM4-64. (bottom) WT cells expressing GFP-BTN1 from its genomic locus and under the control of the BTN1 promoter were grown on glucose-containing medium and were labeled with FM4-64. (B) Btn1 colocalizes with Yif1. WT cells expressing GFP-BTN1 from its genomic locus under the control of a GAL promoter and either RFP-YIF1 from a single-copy plasmid (top row) or SEC7-DsRed from a chromosomal integration (bottom row) were grown on galactose-containing medium and visualized. (C) Btn1 and Btn2 do not colocalize. WT cells expressing GFP-BTN1 from its chromosomal locus under the control of a GAL promoter and BTN2-RFP from a single-copy plasmid are shown; cells were grown on galactose-containing medium. (D) Btn1 is mislocalized to the vacuole in btn2Δ cells. btn2Δ cells expressing GFP-BTN1 from a single-copy plasmid were grown on glucose-containing medium and were labeled with FM4-64. Bars, 1 µm.

Mentions: The intracellular localization of Btn1 is unclear; GFP-tagged Btn1 localizes to the vacuole upon overexpression (i.e., from multicopy plasmids; Croopnick et al., 1998; Pearce and Sherman, 1998, 1999; Pearce et al., 1999; Gachet et al., 2005; Kim et al., 2005) or to the Golgi in S. cerevisiae (Vitiello et al., 2010) and S. pombe (Codlin and Mole, 2009) at lower levels of expression. We examined the localization of Btn1 tagged at the amino terminus with GFP to avoid masking a potential ER export sequence present at the carboxy terminus (position 353–357; LNILE) and expressed it from the genome or single-copy plasmids. GFP-Btn1 expressed from the chromosome under a GAL promoter, constitutively from a single-copy plasmid via an ADH1 promoter, or endogenously under its own promoter localized to numerous small punctate structures that did not colocalize with FM4-64 (Fig. 2 A, top, middle, and bottom rows, respectively). These structures are likely to be trans-Golgi, as GFP-Btn1 colocalized with either RFP-tagged Yif1 or Sec7 (Fig. 2 B), which are both trans-Golgi markers. In contrast, GFP-Btn1 did not overlap with Btn2-RFP (Fig. 2 C), which localizes to LEs (Kama et al., 2007). Thus, in contrast to experiments that show Btn1 to be vacuolar, we observe Btn1 as a Golgi protein. Moreover, GFP-Btn1 localization to the Golgi appears to be mediated by LE–Golgi protein retrieval, as GFP-Btn1 was mislocalized to the vacuole in cells lacking BTN2 (Fig. 2 D), as with Yif1 or Kex2 (Fig. 1, A and C). However, we note that GFP-BTN1 overexpression from multicopy plasmids indeed leads to labeling of the vacuole membrane (unpublished data).


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 localizes to the Golgi in a manner dependent on Btn2. (A) Btn1 does not colocalize with FM4-64–labeled compartments. (top) WT cells expressing GFP-tagged BTN1 from its genomic locus under the control of a GAL promoter (GAL integrated [GAL int]) were grown on galactose-containing medium and were labeled with FM4-64. (middle) WT cells expressing GFP-BTN1 constitutively from a single-copy plasmid (CEN plasmid) were grown on glucose-containing medium and were labeled with FM4-64. (bottom) WT cells expressing GFP-BTN1 from its genomic locus and under the control of the BTN1 promoter were grown on glucose-containing medium and were labeled with FM4-64. (B) Btn1 colocalizes with Yif1. WT cells expressing GFP-BTN1 from its genomic locus under the control of a GAL promoter and either RFP-YIF1 from a single-copy plasmid (top row) or SEC7-DsRed from a chromosomal integration (bottom row) were grown on galactose-containing medium and visualized. (C) Btn1 and Btn2 do not colocalize. WT cells expressing GFP-BTN1 from its chromosomal locus under the control of a GAL promoter and BTN2-RFP from a single-copy plasmid are shown; cells were grown on galactose-containing medium. (D) Btn1 is mislocalized to the vacuole in btn2Δ cells. btn2Δ cells expressing GFP-BTN1 from a single-copy plasmid were grown on glucose-containing medium and were labeled with FM4-64. Bars, 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig2: Btn1 localizes to the Golgi in a manner dependent on Btn2. (A) Btn1 does not colocalize with FM4-64–labeled compartments. (top) WT cells expressing GFP-tagged BTN1 from its genomic locus under the control of a GAL promoter (GAL integrated [GAL int]) were grown on galactose-containing medium and were labeled with FM4-64. (middle) WT cells expressing GFP-BTN1 constitutively from a single-copy plasmid (CEN plasmid) were grown on glucose-containing medium and were labeled with FM4-64. (bottom) WT cells expressing GFP-BTN1 from its genomic locus and under the control of the BTN1 promoter were grown on glucose-containing medium and were labeled with FM4-64. (B) Btn1 colocalizes with Yif1. WT cells expressing GFP-BTN1 from its genomic locus under the control of a GAL promoter and either RFP-YIF1 from a single-copy plasmid (top row) or SEC7-DsRed from a chromosomal integration (bottom row) were grown on galactose-containing medium and visualized. (C) Btn1 and Btn2 do not colocalize. WT cells expressing GFP-BTN1 from its chromosomal locus under the control of a GAL promoter and BTN2-RFP from a single-copy plasmid are shown; cells were grown on galactose-containing medium. (D) Btn1 is mislocalized to the vacuole in btn2Δ cells. btn2Δ cells expressing GFP-BTN1 from a single-copy plasmid were grown on glucose-containing medium and were labeled with FM4-64. Bars, 1 µm.
Mentions: The intracellular localization of Btn1 is unclear; GFP-tagged Btn1 localizes to the vacuole upon overexpression (i.e., from multicopy plasmids; Croopnick et al., 1998; Pearce and Sherman, 1998, 1999; Pearce et al., 1999; Gachet et al., 2005; Kim et al., 2005) or to the Golgi in S. cerevisiae (Vitiello et al., 2010) and S. pombe (Codlin and Mole, 2009) at lower levels of expression. We examined the localization of Btn1 tagged at the amino terminus with GFP to avoid masking a potential ER export sequence present at the carboxy terminus (position 353–357; LNILE) and expressed it from the genome or single-copy plasmids. GFP-Btn1 expressed from the chromosome under a GAL promoter, constitutively from a single-copy plasmid via an ADH1 promoter, or endogenously under its own promoter localized to numerous small punctate structures that did not colocalize with FM4-64 (Fig. 2 A, top, middle, and bottom rows, respectively). These structures are likely to be trans-Golgi, as GFP-Btn1 colocalized with either RFP-tagged Yif1 or Sec7 (Fig. 2 B), which are both trans-Golgi markers. In contrast, GFP-Btn1 did not overlap with Btn2-RFP (Fig. 2 C), which localizes to LEs (Kama et al., 2007). Thus, in contrast to experiments that show Btn1 to be vacuolar, we observe Btn1 as a Golgi protein. Moreover, GFP-Btn1 localization to the Golgi appears to be mediated by LE–Golgi protein retrieval, as GFP-Btn1 was mislocalized to the vacuole in cells lacking BTN2 (Fig. 2 D), as with Yif1 or Kex2 (Fig. 1, A and C). However, we note that GFP-BTN1 overexpression from multicopy plasmids indeed leads to labeling of the vacuole membrane (unpublished data).

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