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A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

Chang FS, Han GS, Carman GM, Blumer KJ - J. Cell Biol. (2005)

Bottom Line: Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility.Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis.Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

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Lsb6 deletion mutants. (A) Schematic of Lsb6 deletion mutants. The two halves of the kinase domains are indicated in gray. The ability of each construct to rescue endosome motility (−, no rescue, +, partial rescue, ++, full rescue; Table I) or interact with Las17 (−, no interaction, +, weak interaction, ++, wild-type interaction, +++, stronger than wild-type interaction; see Fig. 5) is also indicated. (B) Expression of HA-tagged Lsb6 mutant constructs in lsb6Δ cells. The expected sizes of wild-type and mutant forms of HA-tagged Lsb6 in the left panel are: WT, 75 kD; ΔN-terminus, 50 kD; Δkinase subdomain 1, 55 kD; Δlinker, 55 kD; Δkinase subdomain 2, 52 kD; and ΔC-terminus, 60 kD. The expected sizes of the HA-Lsb6 constructs shown in the second panel are: NH2 terminus, 22 kD; kinase subdomain 1, 13 kD; kinase subdomain 1+ linker, 25.7 kD; kinase subdomain 2, 18 kD; COOH terminus, 11.5 kD, and NH2 terminus + kinase subdomain 1, 34 kD. The asterisk indicates a degradation product.
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fig4: Lsb6 deletion mutants. (A) Schematic of Lsb6 deletion mutants. The two halves of the kinase domains are indicated in gray. The ability of each construct to rescue endosome motility (−, no rescue, +, partial rescue, ++, full rescue; Table I) or interact with Las17 (−, no interaction, +, weak interaction, ++, wild-type interaction, +++, stronger than wild-type interaction; see Fig. 5) is also indicated. (B) Expression of HA-tagged Lsb6 mutant constructs in lsb6Δ cells. The expected sizes of wild-type and mutant forms of HA-tagged Lsb6 in the left panel are: WT, 75 kD; ΔN-terminus, 50 kD; Δkinase subdomain 1, 55 kD; Δlinker, 55 kD; Δkinase subdomain 2, 52 kD; and ΔC-terminus, 60 kD. The expected sizes of the HA-Lsb6 constructs shown in the second panel are: NH2 terminus, 22 kD; kinase subdomain 1, 13 kD; kinase subdomain 1+ linker, 25.7 kD; kinase subdomain 2, 18 kD; COOH terminus, 11.5 kD, and NH2 terminus + kinase subdomain 1, 34 kD. The asterisk indicates a degradation product.

Mentions: Accordingly, we generated a series of deletion mutants of HA-Lsb6 expressed from plasmids in lsb6Δ cells (Fig. 4). All Lsb6 deletion constructs exhibited undetectable PI 4-kinase activity (unpublished data). Analysis of endosome motility in lsb6Δ mutants expressing these constructs indicated that the NH2-terminal region flanking the catalytic domain was necessary for endosome motility (Table I). This result is illustrated by comparing Video S9 (endosome motility in an lsb6Δ cell + pHA-Lsb6ΔN-terminus) and Video S7 (endosome motility in an lsb6Δ cell + pLsb6). In contrast, deletion of other regions of Lsb6 did not affect endosome motility (Table I). This result is illustrated by comparing Video S10 (endosome motility in an lsb6Δ cell + pHA-Lsb6ΔC-terminus) and Video S7 (endosome motility in an lsb6Δ cell + pLsb6). Both kinds of results were confirmed quantitatively by using automated particle tracking and MSD plots (Fig. S2). Expression of the NH2-terminal domain of Lsb6 rescued endsome motility less well than a construct containing both the NH2-terminal domain and the first half of the kinase domain (Table I). Taken together, these results indicated that the region of Lsb6 containing the NH2-terminal domain and first half of the kinase domain is necessary and sufficient for full activity.


A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

Chang FS, Han GS, Carman GM, Blumer KJ - J. Cell Biol. (2005)

Lsb6 deletion mutants. (A) Schematic of Lsb6 deletion mutants. The two halves of the kinase domains are indicated in gray. The ability of each construct to rescue endosome motility (−, no rescue, +, partial rescue, ++, full rescue; Table I) or interact with Las17 (−, no interaction, +, weak interaction, ++, wild-type interaction, +++, stronger than wild-type interaction; see Fig. 5) is also indicated. (B) Expression of HA-tagged Lsb6 mutant constructs in lsb6Δ cells. The expected sizes of wild-type and mutant forms of HA-tagged Lsb6 in the left panel are: WT, 75 kD; ΔN-terminus, 50 kD; Δkinase subdomain 1, 55 kD; Δlinker, 55 kD; Δkinase subdomain 2, 52 kD; and ΔC-terminus, 60 kD. The expected sizes of the HA-Lsb6 constructs shown in the second panel are: NH2 terminus, 22 kD; kinase subdomain 1, 13 kD; kinase subdomain 1+ linker, 25.7 kD; kinase subdomain 2, 18 kD; COOH terminus, 11.5 kD, and NH2 terminus + kinase subdomain 1, 34 kD. The asterisk indicates a degradation product.
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fig4: Lsb6 deletion mutants. (A) Schematic of Lsb6 deletion mutants. The two halves of the kinase domains are indicated in gray. The ability of each construct to rescue endosome motility (−, no rescue, +, partial rescue, ++, full rescue; Table I) or interact with Las17 (−, no interaction, +, weak interaction, ++, wild-type interaction, +++, stronger than wild-type interaction; see Fig. 5) is also indicated. (B) Expression of HA-tagged Lsb6 mutant constructs in lsb6Δ cells. The expected sizes of wild-type and mutant forms of HA-tagged Lsb6 in the left panel are: WT, 75 kD; ΔN-terminus, 50 kD; Δkinase subdomain 1, 55 kD; Δlinker, 55 kD; Δkinase subdomain 2, 52 kD; and ΔC-terminus, 60 kD. The expected sizes of the HA-Lsb6 constructs shown in the second panel are: NH2 terminus, 22 kD; kinase subdomain 1, 13 kD; kinase subdomain 1+ linker, 25.7 kD; kinase subdomain 2, 18 kD; COOH terminus, 11.5 kD, and NH2 terminus + kinase subdomain 1, 34 kD. The asterisk indicates a degradation product.
Mentions: Accordingly, we generated a series of deletion mutants of HA-Lsb6 expressed from plasmids in lsb6Δ cells (Fig. 4). All Lsb6 deletion constructs exhibited undetectable PI 4-kinase activity (unpublished data). Analysis of endosome motility in lsb6Δ mutants expressing these constructs indicated that the NH2-terminal region flanking the catalytic domain was necessary for endosome motility (Table I). This result is illustrated by comparing Video S9 (endosome motility in an lsb6Δ cell + pHA-Lsb6ΔN-terminus) and Video S7 (endosome motility in an lsb6Δ cell + pLsb6). In contrast, deletion of other regions of Lsb6 did not affect endosome motility (Table I). This result is illustrated by comparing Video S10 (endosome motility in an lsb6Δ cell + pHA-Lsb6ΔC-terminus) and Video S7 (endosome motility in an lsb6Δ cell + pLsb6). Both kinds of results were confirmed quantitatively by using automated particle tracking and MSD plots (Fig. S2). Expression of the NH2-terminal domain of Lsb6 rescued endsome motility less well than a construct containing both the NH2-terminal domain and the first half of the kinase domain (Table I). Taken together, these results indicated that the region of Lsb6 containing the NH2-terminal domain and first half of the kinase domain is necessary and sufficient for full activity.

Bottom Line: Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility.Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis.Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

Show MeSH
Related in: MedlinePlus