Figure 6. The interaction of CHP3 and mCHP3 with liposomes is regulated by Ca²⁺ and target peptide binding.

Proteins were co-sedimented with POPC:POPS (3:1 molar ratio) liposomes in the presence of either 2 mM Mg²⁺ or 2 mM Mg²⁺ + 2 mM Ca²⁺ at 24°C. Non-myristoylated and myristoylated recoverin (Rec and mRec, respectively) were used as Ca²⁺-myristoyl switch control proteins. (A) Amount of proteins co-sedimented with liposomes was analysed with SDS–PAGE (4–12%, Bis-Tris). (B) Quantification of protein-liposome binding based on the densitometry of bands (SDS–PAGE shown in (A)) corresponded to the co-sedimented proteins with three biological (CHP3 and complexes) or technical (recoverin) replicates. Values were normalized to the respective non-myristoylated protein in the Mg²⁺-bound state. Data are shown as mean ± standard deviation (SD), one-way analysis of variance (ANOVA) with Tukey post-test was performed for mean comparison (statistical significance: n.s. – p > 0.05, **p < 0.01, ***p < 0.001). (C) N-terminal myristoylation of target-associated CHP3 in mouse brain. LC–MS/MS analysis of a trypsin-digested size fraction of solubilized mouse brain membrane containing NHE1-associated CHP3 (see Materials and methods). Upper panel: high coverage of the mouse CHP3 primary sequence by MS/MS-identified peptides (in red; black: sequences not identified, grey: sequences not accessible to our MS analysis) without inclusion of N-myristoyl modification. Lower panel: MS/MS spectrum from the same measurement assigned to the myristoylated tryptic N-terminal peptide of CHP3. Note that no other forms of the N-terminal peptide were detectable in error-tolerant search. Figure 6—source data 1: Full gels for (A) and replicates; raw data of band densitometry and ANOVA test p-values.

Figure 6—figure supplement 1. Sequence coverage and N-terminal myristoylation of endogenous CHP3.

LC–MS/MS analysis of trypsin-digested total membrane fraction obtained from mouse platelets. Upper panel: coverage of the mouse CHP3 primary sequence by MS-identified peptides (in red; black: sequences not identified, grey: sequences not accessible to our MS analysis) without inclusion of N-myristoyl modification. Lower panel: MS/MS spectrum from the same measurement assigned to the myristoylated tryptic N-terminal peptide of CHP3. No other modifications of the N-terminus were detectable in error-tolerant search.