Limits...
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.

Show MeSH

Related in: MedlinePlus

Co-sedimentation of Boi1-PH with vesicles. The two panels show immunoblots, probed with anti-Boi1 antibodies, of supernatant (S) and pellet (P) fractions resulting from centrifugation of a mixture of Boi1-PH and vesicles composed solely of PS (top panel) or composed of a 1:20 (mass:mass) ratio of PIP2 and PS (bottom panel). For this analysis, prior to centrifugation, aliquots of a cytosolic preparation of yeast extract that contained Boi1-PH were incubated with vesicles in PBS buffer that had the following additional concentrations of KCl: 0 (lanes 1 and 2), 50 (lanes 3 and 4), 100 (lanes 5 and 6), and 200 mM (lanes 7 and 8).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC117597&req=5

Figure 3: Co-sedimentation of Boi1-PH with vesicles. The two panels show immunoblots, probed with anti-Boi1 antibodies, of supernatant (S) and pellet (P) fractions resulting from centrifugation of a mixture of Boi1-PH and vesicles composed solely of PS (top panel) or composed of a 1:20 (mass:mass) ratio of PIP2 and PS (bottom panel). For this analysis, prior to centrifugation, aliquots of a cytosolic preparation of yeast extract that contained Boi1-PH were incubated with vesicles in PBS buffer that had the following additional concentrations of KCl: 0 (lanes 1 and 2), 50 (lanes 3 and 4), 100 (lanes 5 and 6), and 200 mM (lanes 7 and 8).

Mentions: To investigate further whether Boi1-PH binds PIP2, we used a vesicle co-sedimentation assay. In this analysis, we asked whether an otherwise soluble fraction of Boi1-PH, after being incubated with PIP2-bearing vesicles, would now sediment in conditions (436,000 × g for 1 hr) that cause vesicles to pellet. (To inhibit the proteolysis of Boi1-PH, we performed this analysis in buffer that lacks Ca++ but that contains EGTA and two other protease inhibitors that were not present in the proteolysis assay.) First, we asked whether Boi1-PH could pellet with vesicles that lacked PIP2. We did not detect any pelleting of Boi1-PH with PC vesicles (data not shown), but found that Boi1-PH could pellet with vesicles composed of PS (Fig. 3, top panel), suggesting that Boi1-PH can bind PS.


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)

Co-sedimentation of Boi1-PH with vesicles. The two panels show immunoblots, probed with anti-Boi1 antibodies, of supernatant (S) and pellet (P) fractions resulting from centrifugation of a mixture of Boi1-PH and vesicles composed solely of PS (top panel) or composed of a 1:20 (mass:mass) ratio of PIP2 and PS (bottom panel). For this analysis, prior to centrifugation, aliquots of a cytosolic preparation of yeast extract that contained Boi1-PH were incubated with vesicles in PBS buffer that had the following additional concentrations of KCl: 0 (lanes 1 and 2), 50 (lanes 3 and 4), 100 (lanes 5 and 6), and 200 mM (lanes 7 and 8).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Co-sedimentation of Boi1-PH with vesicles. The two panels show immunoblots, probed with anti-Boi1 antibodies, of supernatant (S) and pellet (P) fractions resulting from centrifugation of a mixture of Boi1-PH and vesicles composed solely of PS (top panel) or composed of a 1:20 (mass:mass) ratio of PIP2 and PS (bottom panel). For this analysis, prior to centrifugation, aliquots of a cytosolic preparation of yeast extract that contained Boi1-PH were incubated with vesicles in PBS buffer that had the following additional concentrations of KCl: 0 (lanes 1 and 2), 50 (lanes 3 and 4), 100 (lanes 5 and 6), and 200 mM (lanes 7 and 8).
Mentions: To investigate further whether Boi1-PH binds PIP2, we used a vesicle co-sedimentation assay. In this analysis, we asked whether an otherwise soluble fraction of Boi1-PH, after being incubated with PIP2-bearing vesicles, would now sediment in conditions (436,000 × g for 1 hr) that cause vesicles to pellet. (To inhibit the proteolysis of Boi1-PH, we performed this analysis in buffer that lacks Ca++ but that contains EGTA and two other protease inhibitors that were not present in the proteolysis assay.) First, we asked whether Boi1-PH could pellet with vesicles that lacked PIP2. We did not detect any pelleting of Boi1-PH with PC vesicles (data not shown), but found that Boi1-PH could pellet with vesicles composed of PS (Fig. 3, top panel), suggesting that Boi1-PH can bind PS.

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