Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kβ, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinaseβ (PI3Kβ), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.

SummaryRab11 is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. However, the recycling step in which Rab11 participates remains unknown. We show here that, in addition to causing tubulation of recycling endosomes, Rab11 depletion gives rise to accumulation of recycling carriers containing endocytosed transferrin and transferrin receptor beneath the plasma membrane. We also show that the carriers are transported from perinuclear recycling endosomes to the cell periphery along microtubules. Total internal reflection fluorescence microscopy of cells expressing EGFP-tagged transferrin receptor revealed that Rab11 depletion inhibits tethering and fusion of recycling carriers to the plasma membrane. Depletion of Sec15, which interacts with Rab11, or Exo70, both components of the exocyst tethering complex, leads to essentially the same phenotypes as those of Rab11 depletion. Thus, in addition to its role in recycling processes at perinuclear recycling endosomes, Rab11 is transported along microtubules to the cell periphery through association with recycling carriers, and directly regulates vesicle exocytosis at the plasma membrane in concert with the exocyst.

Liprin-␣/SYD-2 is a multimodular scaffolding protein important for presynaptic differentiation and postsynaptic targeting of ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid glutamate receptors. However, the molecular mechanisms underlying these functions remain largely unknown. Here we report that liprin-␣ interacts with the neuron-specific kinesin motor KIF1A. KIF1A colocalizes with liprin-␣ in various subcellular regions of neurons. KIF1A coaccumulates with liprin-␣ in ligated sciatic nerves. KIF1A cofractionates and coimmunopreciptates with liprin-␣ and various liprin-␣-associated membrane, signaling, and scaffolding proteins including ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors, GRIP/ABP, RIM, GIT1, and ␤PIX. These results suggest that liprin-␣ functions as a KIF1A receptor, linking KIF1A to various liprin-␣-associated proteins for their transport in neurons.

Background:The enzymatic activities of mammalian P4-ATPases are incompletely characterized. Results: ATP11A and ATP11C catalyze flipping of NBD-PS and NBD-PE, whereas ATP8B1 preferentially catalyzes flipping of NBD-PC. Furthermore, some PFIC1 mutants of ATP8B1 failed to flip PC. Conclusion: ATP11A/ATP11C and ATP8B1/ATP8B2 preferentially translocate aminophospholipids and PC, respectively. Significance: This is the first evidence showing that the PC-flipping activity of ATP8B1 is associated with the episode of PFIC1.

Liprin-alpha is a multidomain protein that interacts with the LAR family of receptor protein tyrosine phosphatases and the GRIP/ABP family of AMPA receptor-interacting proteins. Previous studies have indicated that liprin-alpha regulates the development of presynaptic active zones and that the association of liprin-alpha with GRIP is required for postsynaptic targeting of AMPA receptors. However, the underlying molecular mechanisms are not well understood. Here we report that liprin-alpha directly interacts with GIT1, a multidomain protein with GTPase-activating protein activity for the ADP-ribosylation factor family of small GTPases known to regulate protein trafficking and the actin cytoskeleton. Electron microscopic analysis indicates that GIT1 distributes to the region of postsynaptic density (PSD) as well as presynaptic active zones. GIT1 is enriched in PSD fractions and forms a complex with liprin-alpha, GRIP, and AMPA receptors in brain. Expression of dominant-negative constructs interfering with the GIT1-liprin-alpha interaction leads to a selective and marked reduction in the dendritic and surface clustering of AMPA receptors in cultured neurons. These results suggest that the GIT1-liprin-alpha interaction is required for AMPA receptor targeting and that GIT1 may play an important role in the organization of presynaptic and postsynaptic multiprotein complexes.