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A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations.

Bentley M, Decker H, Luisi J, Banker G - J. Cell Biol. (2015)

Bottom Line: Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology.We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations.We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jungers Center for Neurosciences Research, Oregon Health & Science University, Portland, OR 97239.

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Identifying the domain of KIF13B that binds to endosomes. (A) As shown in the schematic, different regions of KIF13B tail tagged with FRB were coexpressed with tdTM-BicD2594-FKBP and TfR-GFP. (B) In control cells, TfR-GFP was distributed throughout the cell. After treatment with the linker drug TfR-GFP, vesicles were moved to the cell center in cells expressing FRB-3myc-KIF13B832–1,826 but not FRB-3myc-KIF13B442–831. The yellow lines outline the cell boundaries. Bar, 30 µm.
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fig5: Identifying the domain of KIF13B that binds to endosomes. (A) As shown in the schematic, different regions of KIF13B tail tagged with FRB were coexpressed with tdTM-BicD2594-FKBP and TfR-GFP. (B) In control cells, TfR-GFP was distributed throughout the cell. After treatment with the linker drug TfR-GFP, vesicles were moved to the cell center in cells expressing FRB-3myc-KIF13B832–1,826 but not FRB-3myc-KIF13B442–831. The yellow lines outline the cell boundaries. Bar, 30 µm.

Mentions: In principle, this assay could also be used to define the vesicle-binding domains of trafficking proteins. To evaluate this possibility, we attempted to define the domain of KIF13B that binds early endosomes (Fig. 5). The KIF13B tail contains a forkhead-associated (FHA) domain (residues 423–557) that binds centaurin-α (Tong et al., 2010) and a membrane-associated guanylate kinase (MAGUK) binding stalk (residues 607–831) that interacts with homologues of Drosophila melanogaster discs-large (hDlg; Hanada et al., 2000). Previous studies have implicated the interaction with centaurin-α and the interaction with hDlg as important for the binding of KIF13B to vesicles (Tong et al., 2010; Kanai et al., 2014). We generated an FRB-tagged fragment of the KIF13B tail that contains the FHA domain and the MAGUK binding stalk (KIF13B442–831tail). A second FRB-tagged construct contained the remainder of the tail (KIF13B832–1,826tail). These constructs were each coexpressed with transferrin receptor (TfR)–GFP and FLAG-BicD2594-FKBP (Fig. 5 A). In control cells, TfR-GFP vesicles were distributed throughout the cell (Fig. 5 B). In cells that expressed KIF13B442–831tail and were treated with the linker drug, there was no change in the localization of TfR-GFP vesicles. In cells expressing KIF13B832–1,826tail, addition of the linker drug resulted in a pronounced redistribution of TfR-GFP to the cell center. This experiment shows that residues 832–1,826 are sufficient to mediate binding of KIF13B tail to early endosomes and that neither the FHA domain nor the MAGUK binding stalk mediates this interaction.


A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations.

Bentley M, Decker H, Luisi J, Banker G - J. Cell Biol. (2015)

Identifying the domain of KIF13B that binds to endosomes. (A) As shown in the schematic, different regions of KIF13B tail tagged with FRB were coexpressed with tdTM-BicD2594-FKBP and TfR-GFP. (B) In control cells, TfR-GFP was distributed throughout the cell. After treatment with the linker drug TfR-GFP, vesicles were moved to the cell center in cells expressing FRB-3myc-KIF13B832–1,826 but not FRB-3myc-KIF13B442–831. The yellow lines outline the cell boundaries. Bar, 30 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4315250&req=5

fig5: Identifying the domain of KIF13B that binds to endosomes. (A) As shown in the schematic, different regions of KIF13B tail tagged with FRB were coexpressed with tdTM-BicD2594-FKBP and TfR-GFP. (B) In control cells, TfR-GFP was distributed throughout the cell. After treatment with the linker drug TfR-GFP, vesicles were moved to the cell center in cells expressing FRB-3myc-KIF13B832–1,826 but not FRB-3myc-KIF13B442–831. The yellow lines outline the cell boundaries. Bar, 30 µm.
Mentions: In principle, this assay could also be used to define the vesicle-binding domains of trafficking proteins. To evaluate this possibility, we attempted to define the domain of KIF13B that binds early endosomes (Fig. 5). The KIF13B tail contains a forkhead-associated (FHA) domain (residues 423–557) that binds centaurin-α (Tong et al., 2010) and a membrane-associated guanylate kinase (MAGUK) binding stalk (residues 607–831) that interacts with homologues of Drosophila melanogaster discs-large (hDlg; Hanada et al., 2000). Previous studies have implicated the interaction with centaurin-α and the interaction with hDlg as important for the binding of KIF13B to vesicles (Tong et al., 2010; Kanai et al., 2014). We generated an FRB-tagged fragment of the KIF13B tail that contains the FHA domain and the MAGUK binding stalk (KIF13B442–831tail). A second FRB-tagged construct contained the remainder of the tail (KIF13B832–1,826tail). These constructs were each coexpressed with transferrin receptor (TfR)–GFP and FLAG-BicD2594-FKBP (Fig. 5 A). In control cells, TfR-GFP vesicles were distributed throughout the cell (Fig. 5 B). In cells that expressed KIF13B442–831tail and were treated with the linker drug, there was no change in the localization of TfR-GFP vesicles. In cells expressing KIF13B832–1,826tail, addition of the linker drug resulted in a pronounced redistribution of TfR-GFP to the cell center. This experiment shows that residues 832–1,826 are sufficient to mediate binding of KIF13B tail to early endosomes and that neither the FHA domain nor the MAGUK binding stalk mediates this interaction.

Bottom Line: Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology.We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations.We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jungers Center for Neurosciences Research, Oregon Health & Science University, Portland, OR 97239.

Show MeSH
Related in: MedlinePlus