PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane

Abstract

Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P2. Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.

Fig. 1

A survey of the subcellular localization of 125 small GTPases shows that most PM-localized small GTPases have targeting motifs with clusters of polybasic amino acids. (A) Confocal images of the subcellular localization of CFP-conjugated small GTPases in NIH3T3 cells (full set of images in NIH3T3 and HeLa cells in fig. S1). The four main PM-targeting motifs are represented in the image panels. (B) Correlation between PM localization and the presence of lysine residues in a region 5 to 20 amino acids from the C terminus. (C) Phylogenetic tree of 48 small GTPases that were identified to be partially or fully localized to the PM. Individual membrane targeting elements are color coded: red for polybasic clusters and blue, green, and orange for palmitoyl, prenyl, and myristoyl consensus sequences, respectively. (D) Twenty-amino-acid-long C-terminal tail fragments of Rit and KRas are PM-localized. Lack of PM targeting of a KRas tail fragment without the polybasic region (right). Scale bars, 10 μm.

Fig. 2

Depletion of PI(4,5)P₂ and PI(3,4,5)P₃ dissociates Rit, Rin, and MARCKS ED with polybasic-nonlipid targeting motifs from the PM. (A) Development of a chemically inducible translocation method to deplete PI(4,5)P₂ from the inner leaflet of the PM (22, 23). The PI(4,5)P₂ biosensor YFP-PLCd-PH was cotransfected to monitor depletion of PI(4,5)P₂. (B) Depletion of PI(4,5)P₂ caused only a small reduction in the PM localization of YFP-conjugated Rit tail. (C) A small, but significant, PDGF receptor–mediated increase in Rit tail PM localization can be reversed by addition of the PI3-kinase inhibitor LY29 (before and 9 min after addition of 5 μM iRap). The bar graph shows a quantification of the same experiment. The PM dissociation index is the relative ratio of internal over PM fluorescence; that is, F_1cyt/F_1PM * F_0PM/F_0cyt, with F₀ and F₁ as the fluorescent intensities before and after PI(4,5)P₂ and PI(3,4,5)P₃ depletion. (D) Joint reduction in PI(4,5)P₂ and PI(3,4,5)P₃ triggered a near-complete dissociation of Rit tail and MARCKS ED from the PM. (E) Quantitative analysis of the CF-Inp and/or LY29-triggered PM dissociation of MARCKS ED, and Rit and Rin tails. PLCδ-PH and HRas tails are shown as controls. The inactive LY29 analog LY30 was used as a control (24). Scale bars, 10 μm.

Fig. 3

Polybasic subclusters and hydrophobic amino acids are required for PM targeting by polybasic-nonlipid targeting motifs. (A) Statistical analysis of the relative sequence location of positively charged amino acids in nonprenylated small GTPases. (B) Loss of PM targeting of Rit tail fragment after removal of a single subcluster with positively charged amino acids. (C) Identification of a tryptophan residue in the polybasic regions of Rit and Rin that mediates PM over nuclear localization. Full-length small GTPases, as well as tail fragment mutants, are shown. (D) Identification of two additional hydrophobic amino acid residues that contribute to the PM targeting of Rit. Scale bars, 10 μm.

Fig. 4

Depletion of PI(4,5)P₂ and PI(3,4,5)P₃ dissociates proteins with polybasic-myristoyl and polybasic-prenyl targeting motifs from the PM. (A) Aligned sequences of the N terminus (6 amino acids) and C terminus (20 amino acids) of six ARF family members. Confocal images of an Arl7 mutant construct lacking the N-terminal myristoylation motif (left), a C-terminal tagged Arl7 control construct (middle), and a mutant construct with an internal CFP tag and a flexible C-terminal polybasic Arl7 tail (right). (B) PM localization motifs in geranylgeranylated Rab35. Confocal images from left to right: localization of wild-type Rab35, wild-type tail fragment, wild-type tail lacking geranylgeranylation motif (ΔCC), Rab35 tail ΔCC mutant with KRas caax (Rab35 tail caax), and Rab35 tail caax mutant lacking polybasic amino acids (Rab35 tail caax ΔpB). (C) Depletion of PI(4,5)P₂ by CF-Inp activation without L29 addition causes only a minimal PM dissociation of Arl7, KRas tail fragment, and Rab35. (D) Quantitative analysis of the much larger PM dissociation of polybasic-lipid modified proteins after depletion of PI(4,5)P₂ and PI(3,4,5)P₃ by CF-Inp activation and addition of LY29. Scale bars, 10 μm.