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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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Overexpression or deletion of BTN1 has opposing effects on Golgi SNARE assembly. (A) BTN1 overexpression or deletion affects SNARE partnering with Ykt6. WT and btn1Δ cells expressing myc-tagged Ykt6-1 from a single-copy plasmid and WT cells expressing both myc-tagged Ykt6-1 from a single-copy plasmid and HA-tagged Btn1 from a multicopy plasmid were processed for IP with anti-myc antibodies. Samples of the TCL were separated by SDS-PAGE in parallel to the IP samples. Proteins were detected using antibodies to SNAREs as well as the HA and myc epitopes to detect Btn1 and Ykt6-1, respectively. Note that Ykt6 did not precipitate Sso, Snc, or Btn1. (B) BTN1 overexpression or deletion affects SNARE partnering with Sed5. Control WT and btn1Δ cells expressing HA-Sed5 from a single-copy plasmid and WT cells overexpressing myc-Btn1 from a multicopy plasmid were grown and processed for IP. Samples of the TCL and the IP precipitates were electrophoresed and detected in Western blots, as previously described. Note that Sed5 did not precipitate Tlg2, Sso1/2, or Btn1.
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fig3: Overexpression or deletion of BTN1 has opposing effects on Golgi SNARE assembly. (A) BTN1 overexpression or deletion affects SNARE partnering with Ykt6. WT and btn1Δ cells expressing myc-tagged Ykt6-1 from a single-copy plasmid and WT cells expressing both myc-tagged Ykt6-1 from a single-copy plasmid and HA-tagged Btn1 from a multicopy plasmid were processed for IP with anti-myc antibodies. Samples of the TCL were separated by SDS-PAGE in parallel to the IP samples. Proteins were detected using antibodies to SNAREs as well as the HA and myc epitopes to detect Btn1 and Ykt6-1, respectively. Note that Ykt6 did not precipitate Sso, Snc, or Btn1. (B) BTN1 overexpression or deletion affects SNARE partnering with Sed5. Control WT and btn1Δ cells expressing HA-Sed5 from a single-copy plasmid and WT cells overexpressing myc-Btn1 from a multicopy plasmid were grown and processed for IP. Samples of the TCL and the IP precipitates were electrophoresed and detected in Western blots, as previously described. Note that Sed5 did not precipitate Tlg2, Sso1/2, or Btn1.

Mentions: As Btn1 overproduction impedes the functioning of SNAREs specific to Golgi transport steps (e.g., Ykt6 and Sed5; Fig. S2), we examined SNARE assembly in cells either overexpressing or lacking BTN1. First, we immunoprecipitated an myc epitope–tagged temperature-sensitive form of Ykt6 (e.g., Ykt6-1; see Materials and methods) from WT and either BTN1-overexpressing or btn1Δ cells. In WT cells grown at permissive temperatures, we found that Ykt6-1 readily formed complexes with Sed5 and other SNAREs involved in ER–Golgi transport (e.g., Bet1 and Bos1), intra-Golgi transport (e.g., Sft1 and Gos1), and endosome–Golgi transport (e.g., Tlg1, Tlg2, and Vti1) but not with Sso1/2 or Snc1/2, which act primarily upon exocytosis (Fig. 3 A). In contrast, BTN1 overexpression greatly reduced Ykt6-1 binding to Sed5 as well as to Vti1 and Tlg1 (Fig. 3 A). This indicates that SNARE partners involved in endosome–Golgi transport were less able to form complexes with Ykt6 upon BTN1 up-regulation.


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)

Overexpression or deletion of BTN1 has opposing effects on Golgi SNARE assembly. (A) BTN1 overexpression or deletion affects SNARE partnering with Ykt6. WT and btn1Δ cells expressing myc-tagged Ykt6-1 from a single-copy plasmid and WT cells expressing both myc-tagged Ykt6-1 from a single-copy plasmid and HA-tagged Btn1 from a multicopy plasmid were processed for IP with anti-myc antibodies. Samples of the TCL were separated by SDS-PAGE in parallel to the IP samples. Proteins were detected using antibodies to SNAREs as well as the HA and myc epitopes to detect Btn1 and Ykt6-1, respectively. Note that Ykt6 did not precipitate Sso, Snc, or Btn1. (B) BTN1 overexpression or deletion affects SNARE partnering with Sed5. Control WT and btn1Δ cells expressing HA-Sed5 from a single-copy plasmid and WT cells overexpressing myc-Btn1 from a multicopy plasmid were grown and processed for IP. Samples of the TCL and the IP precipitates were electrophoresed and detected in Western blots, as previously described. Note that Sed5 did not precipitate Tlg2, Sso1/2, or Btn1.
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fig3: Overexpression or deletion of BTN1 has opposing effects on Golgi SNARE assembly. (A) BTN1 overexpression or deletion affects SNARE partnering with Ykt6. WT and btn1Δ cells expressing myc-tagged Ykt6-1 from a single-copy plasmid and WT cells expressing both myc-tagged Ykt6-1 from a single-copy plasmid and HA-tagged Btn1 from a multicopy plasmid were processed for IP with anti-myc antibodies. Samples of the TCL were separated by SDS-PAGE in parallel to the IP samples. Proteins were detected using antibodies to SNAREs as well as the HA and myc epitopes to detect Btn1 and Ykt6-1, respectively. Note that Ykt6 did not precipitate Sso, Snc, or Btn1. (B) BTN1 overexpression or deletion affects SNARE partnering with Sed5. Control WT and btn1Δ cells expressing HA-Sed5 from a single-copy plasmid and WT cells overexpressing myc-Btn1 from a multicopy plasmid were grown and processed for IP. Samples of the TCL and the IP precipitates were electrophoresed and detected in Western blots, as previously described. Note that Sed5 did not precipitate Tlg2, Sso1/2, or Btn1.
Mentions: As Btn1 overproduction impedes the functioning of SNAREs specific to Golgi transport steps (e.g., Ykt6 and Sed5; Fig. S2), we examined SNARE assembly in cells either overexpressing or lacking BTN1. First, we immunoprecipitated an myc epitope–tagged temperature-sensitive form of Ykt6 (e.g., Ykt6-1; see Materials and methods) from WT and either BTN1-overexpressing or btn1Δ cells. In WT cells grown at permissive temperatures, we found that Ykt6-1 readily formed complexes with Sed5 and other SNAREs involved in ER–Golgi transport (e.g., Bet1 and Bos1), intra-Golgi transport (e.g., Sft1 and Gos1), and endosome–Golgi transport (e.g., Tlg1, Tlg2, and Vti1) but not with Sso1/2 or Snc1/2, which act primarily upon exocytosis (Fig. 3 A). In contrast, BTN1 overexpression greatly reduced Ykt6-1 binding to Sed5 as well as to Vti1 and Tlg1 (Fig. 3 A). This indicates that SNARE partners involved in endosome–Golgi transport were less able to form complexes with Ykt6 upon BTN1 up-regulation.

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