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Probing the importance and potential roles of the binding of the PH-domain protein Boi1 to acidic phospholipids.

Hallett MA, Lo HS, Bender A - BMC Cell Biol. (2002)

Bottom Line: Amino-acid substitutions that diminish binding to PIP2 and PS impair localization of Boi1 to the bud, but do not affect the localization of Boi1 to the neck.Conversely, a mutation in the SH3 domain prevents the localization of Boi1 to the neck, but does not impair localization to the bud.Whereas the SH3 domain is needed for localization of Boi1 to the neck, the phospholipid-binding portion of the PH domain is important for localization to the bud.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, IN 47405, USA. hallettm@iupui.edu

ABSTRACT

Background: The related proteins Boi1 and Boi2, which appear to promote polarized growth in S. cerevisiae, both contain a PH (pleckstrin homology) and an SH3 (src homology 3) domain. Previously, we gained evidence that a PH domain-bearing segment of Boi1, which we call Boi1-PH, is sufficient and necessary for function. In the current study, we investigate the binding of Boi1's PH domain to the acidic phospholipids PIP2 (phosphatidylinositol-4,5-bisphosphate) and PS (phosphatidylserine).

Results: Boi1-PH co-sediments with PS vesicles. It does so more readily when these vesicles contain a small amount of PIP2. Boi1-PH is degraded in yeast extracts in a manner that is stimulated by PIP2. Amino-acid substitutions that diminish binding to PIP2 and PS impair Boi1 function. Fusion to a myristoyl group-accepting sequence improves to different degrees the ability of these different mutant versions of Boi1-PH to function. Boi1 and Boi2 are localized to the periphery of buds during much of the budding cycle and to necks late in the cell cycle. Amino-acid substitutions that diminish binding to PIP2 and PS impair localization of Boi1 to the bud, but do not affect the localization of Boi1 to the neck. Conversely, a mutation in the SH3 domain prevents the localization of Boi1 to the neck, but does not impair localization to the bud.

Conclusions: Boi1's PH domain binds to acidic phospholipids, and this binding appears to be important for Boi1 function. The main role of binding to PS may simply be to promote the association of the PH domain with membrane. The higher-affinity binding to PIP2, which apparently promotes a conformational change in the PH domain, may play an important additional role. Boi1 and Boi2 are localized to sites of polarized growth. Whereas the SH3 domain is needed for localization of Boi1 to the neck, the phospholipid-binding portion of the PH domain is important for localization to the bud.

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Effects of different phospholipids on the proteolysis of Boi1-PH. Shown are immunoblots, probed with anti-Boi1 antibodies, of aliquots from a Boi1-PH-bearing yeast extract that was incubated, in the presence of 2 mM CaCl2 (to allow proteolysis), with vesicles containing PS (part A) or PC (part B). The vesicles contained either no additional lipid (lanes 1) or a 1:20 (mass:mass) ratio (with respect to PS or PC) of PI (lanes 2), PI3P (lanes 3), PI4P (lanes 4), or PIP2 (lanes 5).
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Figure 2: Effects of different phospholipids on the proteolysis of Boi1-PH. Shown are immunoblots, probed with anti-Boi1 antibodies, of aliquots from a Boi1-PH-bearing yeast extract that was incubated, in the presence of 2 mM CaCl2 (to allow proteolysis), with vesicles containing PS (part A) or PC (part B). The vesicles contained either no additional lipid (lanes 1) or a 1:20 (mass:mass) ratio (with respect to PS or PC) of PI (lanes 2), PI3P (lanes 3), PI4P (lanes 4), or PIP2 (lanes 5).

Mentions: Fortuitously, we discovered that Boi1-PH can be proteolyzed in yeast extracts in a manner that is stimulated by PIP2. Figure 2A shows an example of this phenomenon; whereas most of the anti-Boi1 immunoreactivity was present in a single band when Boi1-PH was incubated with control PS vesicles that lacked PIP2, almost all of the anti-Boi1 immunoreactivity was present in faster-migrating bands when Boi1-PH was incubated with PS vesicles that contained PIP2 (lanes 1 and 5). (In this and all subsequent experiments that use PIP2/PS mixed vesicles, the mass ratio of PIP2:PS was 1:20.) Less total anti-Boi1 immunoreactivity was recovered when Boi1-PH was incubated with PIP2/PS mixed vesicles than when incubated with vesicles containing only PS (lanes 1 and 5), suggesting that the faster-migrating species represent degradation products of Boi1-PH. Consistent with this view, although Boi1-PH remained stable when incubated for longer intervals with vesicles that contained only PS, the anti-Boi1 immunoreactive bands became fainter and then disappeared altogether when incubated for longer intervals with PIP2/PS mixed vesicles (data not shown).


Probing the importance and potential roles of the binding of the PH-domain protein Boi1 to acidic phospholipids.

Hallett MA, Lo HS, Bender A - BMC Cell Biol. (2002)

Effects of different phospholipids on the proteolysis of Boi1-PH. Shown are immunoblots, probed with anti-Boi1 antibodies, of aliquots from a Boi1-PH-bearing yeast extract that was incubated, in the presence of 2 mM CaCl2 (to allow proteolysis), with vesicles containing PS (part A) or PC (part B). The vesicles contained either no additional lipid (lanes 1) or a 1:20 (mass:mass) ratio (with respect to PS or PC) of PI (lanes 2), PI3P (lanes 3), PI4P (lanes 4), or PIP2 (lanes 5).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117597&req=5

Figure 2: Effects of different phospholipids on the proteolysis of Boi1-PH. Shown are immunoblots, probed with anti-Boi1 antibodies, of aliquots from a Boi1-PH-bearing yeast extract that was incubated, in the presence of 2 mM CaCl2 (to allow proteolysis), with vesicles containing PS (part A) or PC (part B). The vesicles contained either no additional lipid (lanes 1) or a 1:20 (mass:mass) ratio (with respect to PS or PC) of PI (lanes 2), PI3P (lanes 3), PI4P (lanes 4), or PIP2 (lanes 5).
Mentions: Fortuitously, we discovered that Boi1-PH can be proteolyzed in yeast extracts in a manner that is stimulated by PIP2. Figure 2A shows an example of this phenomenon; whereas most of the anti-Boi1 immunoreactivity was present in a single band when Boi1-PH was incubated with control PS vesicles that lacked PIP2, almost all of the anti-Boi1 immunoreactivity was present in faster-migrating bands when Boi1-PH was incubated with PS vesicles that contained PIP2 (lanes 1 and 5). (In this and all subsequent experiments that use PIP2/PS mixed vesicles, the mass ratio of PIP2:PS was 1:20.) Less total anti-Boi1 immunoreactivity was recovered when Boi1-PH was incubated with PIP2/PS mixed vesicles than when incubated with vesicles containing only PS (lanes 1 and 5), suggesting that the faster-migrating species represent degradation products of Boi1-PH. Consistent with this view, although Boi1-PH remained stable when incubated for longer intervals with vesicles that contained only PS, the anti-Boi1 immunoreactive bands became fainter and then disappeared altogether when incubated for longer intervals with PIP2/PS mixed vesicles (data not shown).

Bottom Line: Amino-acid substitutions that diminish binding to PIP2 and PS impair localization of Boi1 to the bud, but do not affect the localization of Boi1 to the neck.Conversely, a mutation in the SH3 domain prevents the localization of Boi1 to the neck, but does not impair localization to the bud.Whereas the SH3 domain is needed for localization of Boi1 to the neck, the phospholipid-binding portion of the PH domain is important for localization to the bud.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, IN 47405, USA. hallettm@iupui.edu

ABSTRACT

Background: The related proteins Boi1 and Boi2, which appear to promote polarized growth in S. cerevisiae, both contain a PH (pleckstrin homology) and an SH3 (src homology 3) domain. Previously, we gained evidence that a PH domain-bearing segment of Boi1, which we call Boi1-PH, is sufficient and necessary for function. In the current study, we investigate the binding of Boi1's PH domain to the acidic phospholipids PIP2 (phosphatidylinositol-4,5-bisphosphate) and PS (phosphatidylserine).

Results: Boi1-PH co-sediments with PS vesicles. It does so more readily when these vesicles contain a small amount of PIP2. Boi1-PH is degraded in yeast extracts in a manner that is stimulated by PIP2. Amino-acid substitutions that diminish binding to PIP2 and PS impair Boi1 function. Fusion to a myristoyl group-accepting sequence improves to different degrees the ability of these different mutant versions of Boi1-PH to function. Boi1 and Boi2 are localized to the periphery of buds during much of the budding cycle and to necks late in the cell cycle. Amino-acid substitutions that diminish binding to PIP2 and PS impair localization of Boi1 to the bud, but do not affect the localization of Boi1 to the neck. Conversely, a mutation in the SH3 domain prevents the localization of Boi1 to the neck, but does not impair localization to the bud.

Conclusions: Boi1's PH domain binds to acidic phospholipids, and this binding appears to be important for Boi1 function. The main role of binding to PS may simply be to promote the association of the PH domain with membrane. The higher-affinity binding to PIP2, which apparently promotes a conformational change in the PH domain, may play an important additional role. Boi1 and Boi2 are localized to sites of polarized growth. Whereas the SH3 domain is needed for localization of Boi1 to the neck, the phospholipid-binding portion of the PH domain is important for localization to the bud.

Show MeSH
Related in: MedlinePlus