Two novel phosphatidylinositol-4-phosphate 5-kinase type Iγ splice variants expressed in human cells display distinctive cellular targeting

The generation of various phosphoinositide messenger molecules at distinct locations within the cell is mediated via the specific targeting of different isoforms and splice variants of phosphoinositide kinases. The lipid messenger PtdIns(4,5)P2 is generated by several of these enzymes when targeted to distinct cellular compartments. Several splice variants of the type Iγ isoform of PIPK (PtdIns4P 5-kinase), which generate PtdIns(4,5)P2, have been identified, and each splice variant is thought to serve a unique functional role within cells. Here, we have identified two novel C-terminal splice variants of PIPKIγ in human cells consisting of 700 and 707 amino acids. These two splice variants are expressed in multiple tissue types and display PIPK activity in vitro. Interestingly, both of these novel splice variants display distinct subcellular targeting. With the addition of these two new splice isoforms, there are minimally five PIPKIγ splice variants that have been identified in mammals. Therefore, we propose the use of the HUGO (Human Genome Organization) nomenclature in the naming of the splice isoforms. PIPKIγ_i4 (700 amino acids) is present in the nucleus, a targeting pattern that has not been previously observed in any PIPKIγ splice variant. PIPKIγ_i5 (707 amino acids) is targeted to intracellular vesicle-like structures, where it co-localizes with markers of several types of endosomal compartments. As occurs with other PIPKIγ splice variants, the distinctive C-terminal sequences of PIPKIγ_i4 and PIPKIγ_i5 may facilitate association with unique protein targeting factors, thereby localizing the kinases to their appropriate cellular subdomains for the site-specific generation of PtdIns(4,5)P2

Receptor Sorting within Endosomal Trafficking Pathway Is Facilitated by Dynamic Actin Filaments

Early endosomes (EEs) are known to be a sorting station for internalized molecules destined for degradation, recycling, or other intracellular organelles. Segregation is an essential step in such sorting, but the molecular mechanism of this process remains to be elucidated. Here, we show that actin is required for efficient recycling and endosomal maturation by producing a motile force. Perturbation of actin dynamics by drugs induced a few enlarged EEs containing several degradative vacuoles and also interfered with their transporting ability. Actin repolymerization induced by washout of the drug caused the vacuoles to dissociate and individually translocate toward the perinuclear region. We further elucidated that cortactin, an actin-nucleating factor, was required for transporting contents from within EEs. Actin filaments regulated by cortactin may provide a motile force for efficient sorting within early endosomes. These data suggest that actin filaments coordinate with microtubules to mediate segregation in EEs

The Cruciality of Single Amino Acid Replacement for the Spectral Tuning of Biliverdin-Binding Cyanobacteriochromes

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms

The Cruciality of Single Amino Acid Replacement for the Spectral Tuning of Biliverdin-Binding Cyanobacteriochromes

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ARAP1 regulates EGF receptor trafficking and signalling

The activation state of the EGF receptor (EGF-R) is tightly controlled in the cell so as to prevent excessive signalling, with the dangerous consequences that this would have on cell growth and proliferation. This control occurs at different levels, with a key level being the trafficking and degradation of the EGF-R itself. Multiple guanosine triphosphatases belonging to the Arf, Rab and Rho families and their accessory proteins have key roles in these processes. In this study, we have identified ARAP1, a multidomain protein with both Arf GTPase-activating protein (GAP) and Rho GAP activities, as a novel component of the machinery that controls the trafficking and signalling of the EGF-R. We show that ARAP1 localizes to multiple cell compartments, including the Golgi complex, as previously reported, and endosomal compartments, where it is enriched in the internal membranes of multivesicular bodies. ARAP1 distribution is controlled by its phosphorylation and by its interactions with the 3- and 4-phosphorylated phosphoinositides through its five PH domains. We provide evidence that ARAP1 controls the late steps of the endocytic trafficking of the EGF-R, with ARAP1 knockdown leading to EGF-R accumulation in a sorting/late endosomal compartment and to the inhibition of EGF-R degradation that is accompanied by prolonged signalling

Sequence-Dependent Sorting of Recycling Proteins by Actin-Stabilized Endosomal Microdomains

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway. © 2010 Elsevier Inc