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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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The deletion of BTN1 results in defects in LE–Golgi sorting. (A) Yif1 is mislocalized to LEs in cells lacking BTN1. (top) WT (BY4741), btn1Δ, and btn2Δ cells expressing GFP-Yif1 from a single-copy plasmid were labeled with FM4-64 and visualized, as described in Materials and methods. Merge indicates merger of the GFP and FM4-64 (i.e., RFP) panels. Light indicates the DIC panel. The third row illustrates endosomal labeling seen with Yif1 in the majority of btn1Δ cells, whereas the fourth row illustrates vacuolar labeling. (bottom) btn1Δ cells expressing GFP-Yif1 from a single-copy plasmid and Cln3 from a multicopy plasmid; cells were labeled as previously described. (B) Yif1 localizes to Vps27-labeled endosomes in btn1Δ cells. WT and btn1Δ cells expressing GFP-Yif1 from a single-copy plasmid and RFP-Vps27 from a multicopy plasmid are shown. (C) Kex2 is mislocalized to LEs in cells lacking BTN1. (top) WT, btn1Δ, and btn2Δ cells expressing Kex2-GFP from a single-copy plasmid are shown. (bottom) btn1Δ cells expressing Kex2-GFP from a single-copy plasmid and Cln3 from a multicopy plasmid are shown. Bars, 1 µm.
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fig1: The deletion of BTN1 results in defects in LE–Golgi sorting. (A) Yif1 is mislocalized to LEs in cells lacking BTN1. (top) WT (BY4741), btn1Δ, and btn2Δ cells expressing GFP-Yif1 from a single-copy plasmid were labeled with FM4-64 and visualized, as described in Materials and methods. Merge indicates merger of the GFP and FM4-64 (i.e., RFP) panels. Light indicates the DIC panel. The third row illustrates endosomal labeling seen with Yif1 in the majority of btn1Δ cells, whereas the fourth row illustrates vacuolar labeling. (bottom) btn1Δ cells expressing GFP-Yif1 from a single-copy plasmid and Cln3 from a multicopy plasmid; cells were labeled as previously described. (B) Yif1 localizes to Vps27-labeled endosomes in btn1Δ cells. WT and btn1Δ cells expressing GFP-Yif1 from a single-copy plasmid and RFP-Vps27 from a multicopy plasmid are shown. (C) Kex2 is mislocalized to LEs in cells lacking BTN1. (top) WT, btn1Δ, and btn2Δ cells expressing Kex2-GFP from a single-copy plasmid are shown. (bottom) btn1Δ cells expressing Kex2-GFP from a single-copy plasmid and Cln3 from a multicopy plasmid are shown. Bars, 1 µm.

Mentions: The loss of BTN2 results in the mislocalization of a trans-Golgi protein, Yif1, to the vacuole (Chattopadhyay et al., 2003; Kama et al., 2007), where it is degraded (Kama et al., 2007). As BTN2 is up-regulated in the absence of BTN1 and because Btn2 acts upon LE–Golgi sorting, we determined whether Btn1 is involved in transport. We examined the localization of GFP-tagged Yif1 in wild-type (WT) cells or cells lacking either BTN1 or BTN2 (Fig. 1 A). In WT cells, GFP-Yif1 labeled small punctate structures that correspond to Golgi (Matern et al., 2000) and did not colabel with FM4-64 (Fig. 1 A and Table S1), a dye that labels endosomes and then vacuoles. In contrast, GFP-Yif1 mislocalized to vacuoles in btn2Δ cells, as previously shown (Chattopadhyay et al., 2003; Kama et al., 2007), and to FM4-64–labeled compartments situated adjacent to the vacuole (along with limited vacuolar labeling) in btn1Δ cells (Fig. 1 A and Table S1). The nonvacuolar compartments labeled by GFP-Yif1 in btn1Δ cells are likely to be LEs, as they colabeled with RFP-tagged Vps27 (Fig. 1 B), a protein involved in the multivesicular body (MVB) pathway (Piper et al., 1995). Thus, the deletion of BTN1 affects Yif1 localization in a manner similar to the deletion of BTN2. Importantly, Yif1 localization to the Golgi was almost fully restored to btn1Δ cells by expression of the human CLN3 gene (Fig. 1 A [bottom] and Table S1). Thus, the function of these orthologues may be conserved.


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)

The deletion of BTN1 results in defects in LE–Golgi sorting. (A) Yif1 is mislocalized to LEs in cells lacking BTN1. (top) WT (BY4741), btn1Δ, and btn2Δ cells expressing GFP-Yif1 from a single-copy plasmid were labeled with FM4-64 and visualized, as described in Materials and methods. Merge indicates merger of the GFP and FM4-64 (i.e., RFP) panels. Light indicates the DIC panel. The third row illustrates endosomal labeling seen with Yif1 in the majority of btn1Δ cells, whereas the fourth row illustrates vacuolar labeling. (bottom) btn1Δ cells expressing GFP-Yif1 from a single-copy plasmid and Cln3 from a multicopy plasmid; cells were labeled as previously described. (B) Yif1 localizes to Vps27-labeled endosomes in btn1Δ cells. WT and btn1Δ cells expressing GFP-Yif1 from a single-copy plasmid and RFP-Vps27 from a multicopy plasmid are shown. (C) Kex2 is mislocalized to LEs in cells lacking BTN1. (top) WT, btn1Δ, and btn2Δ cells expressing Kex2-GFP from a single-copy plasmid are shown. (bottom) btn1Δ cells expressing Kex2-GFP from a single-copy plasmid and Cln3 from a multicopy plasmid are shown. Bars, 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig1: The deletion of BTN1 results in defects in LE–Golgi sorting. (A) Yif1 is mislocalized to LEs in cells lacking BTN1. (top) WT (BY4741), btn1Δ, and btn2Δ cells expressing GFP-Yif1 from a single-copy plasmid were labeled with FM4-64 and visualized, as described in Materials and methods. Merge indicates merger of the GFP and FM4-64 (i.e., RFP) panels. Light indicates the DIC panel. The third row illustrates endosomal labeling seen with Yif1 in the majority of btn1Δ cells, whereas the fourth row illustrates vacuolar labeling. (bottom) btn1Δ cells expressing GFP-Yif1 from a single-copy plasmid and Cln3 from a multicopy plasmid; cells were labeled as previously described. (B) Yif1 localizes to Vps27-labeled endosomes in btn1Δ cells. WT and btn1Δ cells expressing GFP-Yif1 from a single-copy plasmid and RFP-Vps27 from a multicopy plasmid are shown. (C) Kex2 is mislocalized to LEs in cells lacking BTN1. (top) WT, btn1Δ, and btn2Δ cells expressing Kex2-GFP from a single-copy plasmid are shown. (bottom) btn1Δ cells expressing Kex2-GFP from a single-copy plasmid and Cln3 from a multicopy plasmid are shown. Bars, 1 µm.
Mentions: The loss of BTN2 results in the mislocalization of a trans-Golgi protein, Yif1, to the vacuole (Chattopadhyay et al., 2003; Kama et al., 2007), where it is degraded (Kama et al., 2007). As BTN2 is up-regulated in the absence of BTN1 and because Btn2 acts upon LE–Golgi sorting, we determined whether Btn1 is involved in transport. We examined the localization of GFP-tagged Yif1 in wild-type (WT) cells or cells lacking either BTN1 or BTN2 (Fig. 1 A). In WT cells, GFP-Yif1 labeled small punctate structures that correspond to Golgi (Matern et al., 2000) and did not colabel with FM4-64 (Fig. 1 A and Table S1), a dye that labels endosomes and then vacuoles. In contrast, GFP-Yif1 mislocalized to vacuoles in btn2Δ cells, as previously shown (Chattopadhyay et al., 2003; Kama et al., 2007), and to FM4-64–labeled compartments situated adjacent to the vacuole (along with limited vacuolar labeling) in btn1Δ cells (Fig. 1 A and Table S1). The nonvacuolar compartments labeled by GFP-Yif1 in btn1Δ cells are likely to be LEs, as they colabeled with RFP-tagged Vps27 (Fig. 1 B), a protein involved in the multivesicular body (MVB) pathway (Piper et al., 1995). Thus, the deletion of BTN1 affects Yif1 localization in a manner similar to the deletion of BTN2. Importantly, Yif1 localization to the Golgi was almost fully restored to btn1Δ cells by expression of the human CLN3 gene (Fig. 1 A [bottom] and Table S1). Thus, the function of these orthologues may be conserved.

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