PTEN phosphatase selectively binds phosphoinositides and undergoes structural changes

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor that is mutated or deleted in a variety of human tumors, and even loss of only one PTEN gene profoundly affects carcinogenesis. PTEN encodes a phosphatidylinositol phosphate phosphatase specific for the 3-position of the inositol ring. Despite its importance, we are just beginning to understand the regulatory circuits that maintain the correct levels of PTEN phosphatase activity. Several independent studies reported that PI(4,5)P2 enhances PTEN phosphatase activity, but the reasons for this enhancement are currently being debated. In this study, PTEN bound to PI(4,5)P2-bearing vesicles has increased alpha-helicity, providing direct spectroscopic proof of a conformational change. Neither PI(3,5)P2 nor PI(3,4,5)P3 induced this conformational change. On the basis of experiments with two mutant PTEN proteins, it is shown that PI(4,5)P2 induces this conformational change by binding to the PTEN N-terminal domain. Using PTEN protein and a 21-amino acid peptide based on the PTEN N-terminus, we tested all natural phosphatidylinositol phosphates and found preferential binding of PI(4,5)P2. PTEN also binds to phosphatidylserine-bearing vesicles, resulting in a slight increase in beta-sheet content. In addition, PTEN binds synergistically to PI(4,5)P2 and phosphatidylserine, and hence, these anionic lipids do not compete for PTEN binding sites. Collectively, these results demonstrate that PTEN binds to membranes through multiple sites, but only PI(4,5)P2 binding to the N-terminal domain triggers a conformational change with increased alpha-helicity.     

Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p

Vesicles in eukaryotic cells transport cargo between functionally distinct membrane-bound organelles and the plasma membrane for growth and secretion. Trafficking and fusion of vesicles to specific target sites are highly regulated processes that are not well understood at the molecular level. At the plasma membrane, tethering and fusion of secretory vesicles require the exocyst complex. As a step toward elucidation of the molecular architecture and biochemical function(s) of the exocyst complex, we expressed and purified the exocyst subunit Sec6p and demonstrated that it is a predominantly helical protein. Biophysical characterization of purified Sec6p by gel filtration and analytical ultracentrifugation experiments revealed that Sec6p is a dimer. Limited proteolysis defined an independently folded C-terminal domain (residues 300-805) that equilibrated between a dimer and monomer in solution. Removal of residues 300-410 from this construct yielded a well-folded, monomeric domain. These results demonstrate that residues 300-410 are necessary for dimerization, and the presence of the N-terminal region (1-299) increases dimer stability. Moreover, we found that the dimer of Sec6p binds to the plasma membrane t-SNARE Sec9p and inhibits the interaction between Sec9p and its partner t-SNARE Sso1p. This direct interaction between the exocyst complex and the t-SNARE implicates the exocyst in SNARE complex regulation.     

The structure of the exocyst subunit Sec6p defines a conserved architecture with diverse roles

The exocyst is a conserved protein complex essential for trafficking secretory vesicles to the plasma membrane. The structure of the C-terminal domain of the exocyst subunit Sec6p reveals multiple helical bundles, which are structurally and topologically similar to Exo70p and the C-terminal domains of Exo84p and Sec15, despite <10% sequence identity. The helical bundles appear to be evolutionarily related molecular scaffolds that have diverged to create functionally distinct exocyst proteins.