Rohan D. Teasdale

Oligomeric Complexes Link Rab5 Effectors with NSF and Drive Membrane Fusion via Interactions between EEA1 and Syntaxin 13

SNAREs and Rab GTPases cooperate in vesicle transport through a mechanism yet poorly understood. We now demonstrate that the Rab5 effectors EEA1 and Rabaptin-5/Rabex-5 exist on the membrane in high molecular weight oligomers, which also contain NSF. Oligomeric assembly is modulated by the ATPase activity of NSF. Syntaxin 13, the t-SNARE required for endosome fusion, is transiently incorporated into the large oligomers via direct interactions with EEA1. This interaction is required to drive fusion, since both dominant-negative EEA1 and synthetic peptides encoding the FYVE Zn2+ finger hinder the interaction and block fusion. We propose a novel mechanism whereby oligomeric EEA1 and NSF mediate the local activation of syntaxin 13 upon membrane tethering and, by analogy with viral fusion proteins, coordinate the assembly of a fusion pore.

Twenty pairs of sox: extent, homology, and nomenclature of the mouse and human sox transcription factor gene families.

We thank Dr. Elspeth Bruford, HUGO Nomenclature Committee, for comments on the manuscript and helpful discussions. We apologize to colleagues whose work was not cited directly due to space constraints. P.K. is an Australian Research Council Professorial Research Fellow.

Human pigmentation genes: identification, structure and consequences of polymorphic variation

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones alpha-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation.

Letter to the editorTwenty Pairs of Sox: Extent, Homology, and Nomenclature of the Mouse and Human Sox Transcription Factor Gene Families

We thank Dr. Elspeth Bruford, HUGO Nomenclature Committee, for comments on the manuscript and helpful discussions. We apologize to colleagues whose work was not cited directly due to space constraints. P.K. is an Australian Research Council Professorial Research Fellow.

Prediction of protein B-factor profiles

The polypeptide backbones and side chains of proteins are constantly moving due to thermal motion and the kinetic energy of the atoms. The B‐factors of protein crystal structures reflect the fluctuation of atoms about their average positions and provide important information about protein dynamics. Computational approaches to predict thermal motion are useful for analyzing the dynamic properties of proteins with unknown structures. In this article, we utilize a novel support vector regression (SVR) approach to predict the B‐factor distribution (B‐factor profile) of a protein from its sequence. We explore schemes for encoding sequences and various settings for the parameters used in SVR. Based on a large dataset of high‐resolution proteins, our method predicts the B‐factor distribution with a Pearson correlation coefficient (CC) of 0.53. In addition, our method predicts the B‐factor profile with a CC of at least 0.56 for more than half of the proteins. Our method also performs well for classifying residues (rigid vs. flexible). For almost all predicted B‐factor thresholds, prediction accuracies (percent of correctly predicted residues) are greater than 70%. These results exceed the best results of other sequence‐based prediction methods. Proteins 2005.

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

The Phox Homology (PX) Domain-dependent, 3-Phosphoinositide-mediated Association of Sorting Nexin-1 with an Early Sorting Endosomal Compartment Is Required for Its Ability to Regulate Epidermal Growth Factor Receptor Degradation

Recent studies have shown that phox homology (PX) domains act as phosphoinositide-binding motifs. The majority of PX domains studied show binding to phosphatidylinositol 3-monophosphate (PtdIns(3)P), an association that allows the host protein to localize to membranes of the endocytic pathway. One issue, however, is whether PX domains may have alternative phosphoinositide binding specificities that could target their host protein to distinct subcellular compartments or allow their allosteric regulation by phosphoinositides other than PtdIns(3)P. It has been reported that the PX domain of sorting nexin 1 (SNX1) specifically binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) (Zhong, Q., Lazar, C. S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S. D., and Gill, G. N. (2002)Proc. Natl. Acad. Sci. U. S. A. 99, 6767–6772). In the present study, we have shown that whereas SNX1 binds PtdIns(3,4,5)P3 in protein:lipid overlay assays, in liposomes-based assays, binding is observed to PtdIns(3)P and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) but not to PtdIns(3,4,5)P3. To address the significance of PtdIns(3,4,5)P3 binding, we examined the subcellular localization of SNX1 under conditions in which plasma membrane PtdIns(3,4,5)P3 levels were significantly elevated. Under these conditions, we failed to observe association of SNX1 with this membrane. However, consistent with the binding to PtdIns(3)P and PtdIns(3,5)P2 being of more physiological significance was the observation that the association of SNX1 with an early endosomal compartment was dependent on a 3-phosphoinositide-binding PX domain and the presence of PtdIns(3)P on this compartment. Finally, we have shown that the PX domain-dependent/early endosomal association of SNX1 is important for its ability to regulate the targeting of internalized epidermal growth factor receptor for lysosomal degradation.

Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions and roles in disease.

The mammalian genome encodes 49 proteins that possess a PX (phox-homology) domain, responsible for membrane attachment to organelles of the secretory and endocytic system via binding of phosphoinositide lipids. The PX domain proteins, most of which are classified as SNXs (sorting nexins), constitute an extremely diverse family of molecules that play varied roles in membrane trafficking, cell signalling, membrane remodelling and organelle motility. In the present review, we present an overview of the family, incorporating recent functional and structural insights, and propose an updated classification of the proteins into distinct subfamilies on the basis of these insights. Almost all PX domain proteins bind PtdIns3P and are recruited to early endosomal membranes. Although other specificities and localizations have been reported for a select few family members, the molecular basis for binding to other lipids is still not clear. The PX domain is also emerging as an important protein-protein interaction domain, binding endocytic and exocytic machinery, transmembrane proteins and many other molecules. A comprehensive survey of the molecular interactions governed by PX proteins highlights the functional diversity of the family as trafficking cargo adaptors and membrane-associated scaffolds regulating cell signalling. Finally, we examine the mounting evidence linking PX proteins to different disorders, in particular focusing on their emerging importance in both pathogen invasion and amyloid production in Alzheimers disease.

EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin.

In epithelia, junction proteins are endocytosed for modulation of cell-cell adhesion and cell polarity. In response to growth factors, the cell-cell adhesion protein E-cadherin is internalized from the cell surface with degradation or recycling as potential fates. However, the cellular machinery involved in cadherin internalization and recycling remains controversial. Here we investigated EGF-induced E-cadherin internalization. EGF stimulation of MCF-7 cells resulted in Rac1-modulated macropinocytosis of the E-cadherin-catenin complex into endosomal compartments that colocalized with EEA1 and the sorting nexin, SNX1. Depletion of cellular SNX1 levels by siRNA resulted in increased intracellular accumulation and turnover of E-cadherin internalized from the cell surface in response to EGF. Moreover, SNX1 was also required for efficient recycling of internalized E-cadherin and re-establishment of epithelial adhesion. Together, these findings demonstrate a role for SNX1 in retrieval of E-cadherin from a degradative endosomal pathway and in membrane trafficking pathways that regulate E-cadherin recycling.

In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts.

ABSTRACT The growth hormone receptor (GHR) is a critical regulator of postnatal growth and metabolism. However, the GHR signaling domains and pathways that regulate these processes in vivo are not defined. We report the first knock-in mouse models with deletions of specific domains of the receptor that are required for its in vivo actions. Mice expressing truncations at residue m569 (plus Y539/545-F) and at residue m391 displayed a progressive impairment of postnatal growth with receptor truncation. Moreover, after 4 months of age, marked male obesity was observed in both mutant 569 and mutant 391 and was associated with hyperglycemia. Both mutants activated hepatic JAK2 and ERK2, whereas STAT5 phosphorylation was substantially decreased for mutant 569 and absent from mutant 391, correlating with loss of IGF-1 expression and reduction in growth. Microarray analysis of these and GHR−/− mice demonstrated that particular signaling domains are responsible for the regulation of different target genes and revealed novel actions of growth hormone. These mice represent the first step in delineating the domains of the GHR regulating body growth and composition and the transcripts associated with these domains.