Pleckstrin homology (PH) domains are small protein modules known for their ability to bind phosphoinositides and to drive membrane recruitment of their host proteins. We investigated phosphoinositide binding (in vitro and in vivo) and subcellular localization, and we modeled the electrostatic properties for all 33 PH domains encoded in the S. cerevisiae genome. Only one PH domain (from Num1p) binds phosphoinositides with high affinity and specificity. Six bind phosphoinositides with moderate affinity and little specificity and are membrane targeted in a phosphoinositide-dependent manner. Although all of the remaining 26 yeast PH domains bind phosphoinositides very weakly or not at all, three were nonetheless efficiently membrane targeted. Our proteome-wide analysis argues that membrane targeting is important for only approximately 30% of yeast PH domains and is defined by binding to both phosphoinositides and other targets. These findings have significant implications for understanding the function of proteins that contain this common domain.

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Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca2'-induced conformational changes in troponin C (TnC). Rabbit skeletal TnC contains two tyrosines (YlO in the N-helix, and Y109 in site 3 in the C-terminal domain) in distinct microenviromnents: their individual contributions to total fluorescence intensity are elucidated here utilizing bacterially synthesized rabbit skeletal TnC (sTnC4) and a genetically engineered variant, termed 109YF, lacking one of the tyrosines (Y109 replaced with F109). The steady-state fluorescence emission spectra following excitation at 280 nm were recorded in EGTA (Cat'-free) and Ca*'-saturated (pCa4) solutions. For the wild-type sTnC4, pCa4 causes a significant (46%) increase in the peak fluorescence intensity over the value in EGTA. For the mutant 109YF, the EGTA fluorescence is only marginally affected (74% of the wild-type Fao&, but interestingly the Ca2' effect is completely suppressed (AF = Fe, -&.xA = 2% of the wild-type value). These results indicate that the two tyrosines make disparate contributions to the fluorescence spectrum of wild-type sTnC, both in the presence and absence of Ca'+; whereas Y 10 in the N-helix is dominant in Ca*'-free solution, Y 109 is the sole contributor to the Ca*+ effect. Furthermore, to explain the biphasic fluorescence response of Y 109 obtained during Ca*+ titrations, the hndings yield the most unequivocal evidence that Ca*+-induced conformational changes in the trigger sites operating the contractile switch modify properties of the C-terminal sites in TnC pari passu.

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