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Linda Hicke

Publications and source records attributed to Linda Hicke.

15 recordsLinked to original sources
PubMed2006

Structural basis for monoubiquitin recognition by the Ede1 UBA domain.

Monoubiquitination is a general mechanism for downregulating the activity of cell surface receptors by consigning these proteins for lysosome-mediated degradation through the endocytic pathway. The yeast Ede1 protein functions at the internalization step of endocytosis and binds monoubiquitinated proteins through a ubiquitin associated (UBA) domain. UBA domains are found in a broad range of cellular proteins but previous studies have suggested that the mode of ubiquitin recognition might not be universally conserved. Here we present the solution structure of the Ede1 UBA domain in complex with monoubiquitin. The Ede1 UBA domain forms a three-helix bundle structure and binds ubiquitin through a largely hydrophobic surface in a manner reminiscent of the Dsk2 UBA and the remotely homologous Cue2 CUE domains, for which high-resolution structures have been described. However, the interaction is dissimilar to the molecular models proposed for the hHR23A UBA domains bound to either monoubiquitin or Lys48-linked diubiquitin. Our mutational analyses of the Ede1 UBA domain-ubiquitin interaction reveal several key affinity determinants and, unexpectedly, a negative affinity determinant in the wild-type Ede1 protein, implying that high-affinity interactions may not be the sole criterion for optimal function of monoubiquitin-binding endocytic proteins.

Amino Acid Sequence↗
PubMed2005

Identification and characterization of modular domains that bind ubiquitin.

To receive and transmit the information carried by ubiquitin signals, cells have evolved an array of modular ubiquitin-binding domains. These domains bind directly and noncovalently to monoubiquitin and polyubiquitin chains and are found within proteins that function in diverse biological processes. Ubiquitin-binding domains characterized thus far are generally small and structurally diverse, yet they all interact with the same hydrophobic patch on the surface of ubiquitin. The rapid identification and characterization of ubiquitin-binding domains has been accomplished through the extensive use of bioinformatics, biochemistry, molecular biology, and biophysics. Here, we discuss the strategies and tools that have been most successful in the identification and characterization of ubiquitin-binding domains.

Chromatography, Affinity↗
PubMed2005

Ubiquitin-binding domains.

Ubiquitin-binding domains (UBDs) are a collection of modular protein domains that non-covalently bind to ubiquitin. These recently discovered motifs interpret and transmit information conferred by protein ubiquitylation to control various cellular events. Detailed molecular structures are known for a number of UBDs, but to understand their mechanism of action, we also need to know how binding specificity is determined, how ubiquitin binding is regulated, and the function of UBDs in the context of full-length proteins. Such knowledge will be key to our understanding of how ubiquitin regulates cellular proteins and processes.

Carrier Proteins↗
PubMed2004

The Rsp5 ubiquitin ligase binds to and ubiquitinates members of the yeast CIN85-endophilin complex, Sla1-Rvs167.

Sla1 and Rvs167 are yeast proteins required for receptor internalization and organization of the actin cytoskeleton. Here we provide evidence that Sla1 and Rvs167 are orthologues of the mammalian CIN85 and endophilin proteins, respectively, which are required for ligand-stimulated growth factor receptor internalization. Sla1 is similar in domain structure to CIN85 and binds directly to the endophilin-like Rvs167. Akin to CIN85, Sla1 interacts with synaptojanins and a ubiquitin ligase that regulates endocytosis. This ubiquitin ligase, Rsp5, binds directly to both Sla1 and Rvs167. The interaction between Rsp5 and Rvs167 is mediated through Rsp5 WW domains and PXY motifs in the central Gly-Pro-Ala-rich domain of Rvs167. Rvs167 PXY motifs are required for Rsp5-dependent monoubiquitination of Rvs167 on Lys481 in the Src homology 3 (SH3) domain. Mutation of Lys481 --> Arg causes cells to grow slowly on medium containing 1 M NaCl, although this phenotype is not due to the defect in ubiquitination caused by the K481R mutation. We propose that Rsp5 interaction with Sla1-Rvs167 promotes Rvs167 ubiquitination and regulates activity of this protein complex. Rvs167 ubiquitination is not required for general function of Rvs167, but may control specific Rvs167 SH3 domain-protein interactions or negatively regulate SH3 domain activity.

Amino Acid Sequence↗
PubMed2004

The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo.

Ubiquitin ligases of the Nedd4 family regulate membrane protein trafficking by modifying both cargo proteins and the transport machinery with ubiquitin. Here, we investigate the role of the yeast Nedd4 homologue, Rsp5, in protein sorting into vesicles that bud into the multivesicular endosome (MVE) en route to the vacuole. A mutant lacking the Rsp5 C2 domain is unable to ubiquitinate or sort biosynthetic cargo into MVE vesicles, whereas endocytic cargo is ubiquitinated and sorted efficiently. The C2 domain binds specifically to phosphoinositides in vitro and is sufficient for localization to membranes in intact cells. Mutation of a lysine-rich patch on the surface of the C2 domain abolishes membrane interaction and disrupts sorting of biosynthetic cargo. Translational fusion of ubiquitin to a biosynthetic cargo protein alleviates the requirement for the C2 domain in its MVE sorting. These results demonstrate that the C2 domain specifies Rsp5-dependent ubiquitination of endosomal cargo and suggest that Rsp5 function is regulated by membrane phosphoinositides.

Carrier Proteins↗
PubMed2004

Ubiquitin-dependent regulation of the synapse.

Posttranslational modification of cellular proteins by the covalent attachment of ubiquitin regulates protein stability, activity, and localization. Ubiquitination is rapid and reversible and is a potent mechanism for the spatial and temporal control of protein activity. By sculpting the molecular composition of the synapse, this versatile posttranslational modification shapes the pattern, activity, and plasticity of synaptic connections. Synaptic processes regulated by ubiquitination, as well as ubiquitination enzymes and their targets at the synapse, are being identified by genetic, biochemical, and electrophysiological analyses. This work provides tantalizing hints that neuronal activity collaborates with ubiquitination pathways to regulate the structure and function of synapses.

Animals↗
PubMed2003

Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation.

Monoubiquitylation is a well-characterized signal for the internalization and sorting of integral membrane proteins to distinct cellular organelles. Recognition and transmission of monoubiquitin signals is mediated by a variety of ubiquitin-binding motifs such as UIM, UBA, UEV, VHS and CUE in endocytic proteins. The yeast Vps27 protein requires two UIMs for efficient interactions with ubiquitin and for sorting cargo into multivesicular bodies. Here we show that the individual UIMs of Vps27 exist as autonomously folded alpha-helices that bind ubiquitin independently, non-cooperatively and with modest affinity. The Vps27 N-terminal UIM engages the Leu8-Ile44-Val70 hydrophobic patch of ubiquitin through a helical surface conserved in UIMs of diverse proteins, including that of the S5a proteasomal regulatory subunit. The Leu8-Ile44-Val70 ubiquitin surface is also the site of interaction for CUE and UBA domains in endocytic proteins, consistent with the view that ubiquitin-binding endocytic proteins act serially on the same monoubiquitylated cargo during transport from cell surface to the lysosome.

Amino Acid Sequence↗
PubMed2003

Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding.

Monoubiquitination serves as a regulatory signal in a variety of cellular processes. Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs. Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination. The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin. The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation. This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling. The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch. Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin. The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins.

Amino Acid Motifs↗
PubMed2003

A ubiquitin-binding motif required for intramolecular monoubiquitylation, the CUE domain.

Monoubiquitylation is a regulatory signal, like phosphorylation, that can alter the activity, location or structure of a protein. Monoubiquitin signals are likely to be recognized by ubiquitin-binding proteins that transmit the regulatory information conferred by monoubiquitylation. To identify monoubiquitin-binding proteins, we used a mutant ubiquitin that lacks the primary site of polyubiquitin chain formation as bait in a two-hybrid screen. The C-terminus of Vps9, a protein required in the yeast endocytic pathway, interacted specifically with monoubiquitin. The region required for monoubiquitin binding mapped to the Vps9 CUE domain, a sequence previously identified by database searches as similar to parts of the yeast Cue1 and mammalian Tollip proteins. We demonstrate that CUE domains bind directly to monoubiquitin and we have defined crucial interaction surfaces on both binding partners. The Vps9 CUE domain is required to promote monoubiquitylation of Vps9 by the Rsp5 hect domain ubiquitin ligase. Thus, we conclude that the CUE motif is an evolutionarily conserved monoubiquitin-binding domain that mediates intramolecular monoubiquitylation.

Amino Acid Motifs↗
PubMed2003

PtdIns(3,5)P2 finds a partner.

Phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P(2)) is required for the sorting of a subset of membrane proteins at the late endosome. Unlike other phosphoinositides, binding partners for PtdIns(3,5)P(2) and its mechanism of action have not been characterized. New work by in this issue of Developmental Cell describes the identification of a yeast epsin-like protein that binds PtdIns(3,5)P(2) and functions in the transport of proteins through late endosomes to the lysosome-like vacuole.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

Receptor internalization in yeast requires the Tor2-Rho1 signaling pathway.

Efficient internalization of proteins from the cell surface is essential for regulating cell growth and differentiation. In a screen for yeast mutants defective in ligand-stimulated internalization of the alpha-factor receptor, we identified a mutant allele of TOR2, tor2G2128R. Tor proteins are known to function in translation initiation and nutrient sensing and are required for cell cycle progression through G1. Yeast Tor2 has an additional role in regulating the integrity of the cell wall by activating the Rho1 guanine nucleotide exchange factor Rom2. The endocytic defect in tor2G2128R cells is due to disruption of this Tor2 unique function. Other proteins important for cell integrity, Rom2 and the cell integrity sensor Wsc1, are also required for efficient endocytosis. A rho1 mutant specifically defective in activation of the glucan synthase Fks1/2 does not internalize alpha-factor efficiently, and fks1Delta cells exhibit a similar phenotype. Removal of the cell wall does not inhibit internalization, suggesting that the function of Rho1 and Fks1 in endocytosis is not through cell wall synthesis or structural integrity. These findings reveal a novel function for the Tor2-Rho1 pathway in controlling endocytosis in yeast, a function that is mediated in part through the plasma membrane protein Fks1.

Cell Cycle Proteins↗
PubMed2003

Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins.

Ubiquitin regulates protein transport between membrane compartments by serving as a sorting signal on protein cargo and by controlling the activity of trafficking machinery. Monoubiquitin attached to integral plasma membrane proteins or to associated transport modifiers serves as a regulated signal for internalization into the endocytic pathway. Similarly, monoubiquitin attached to biosynthetic and endocytic membrane proteins is a signal for sorting of cargo into vesicles that bud into the late endosome lumen for delivery into the lysosome. Ubiquitination of trans-acting endocytic proteins is also required for transport, and key endocytic proteins are modified by monoubiquitin. Regulatory enzymes of the ubiquitination machinery, ubiquitin ligases, control the timing and specificity of plasma membrane protein downregulation in such diverse biological processes as cell fate specification and neurotransmission. Monoubiquitin signals appended by these ligases are recognized by endocytic proteins carrying ubiquitin-binding motifs, including UBA, UEV, UIM, and CUE domains. The UIM proteins epsins and Hrs are excellent candidates for adaptors that link ubiquitinated cargo to the clathrin-based sorting machinery at appropriate regions of the endosomal or plasma membranes. Other ubiquitin-binding proteins also play crucial roles in cargo transport, although in most cases the role of ubiquitin-binding is not defined. Ubiquitin-binding proteins such as epsins, Hrs, and Vps9 are monoubiquitinated, indicating the general nature of ubiquitin regulation in endocytosis and suggesting new models to explain how recognition of monoubiquitin signals may be regulated.

Animals↗
PubMed2002

The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast.

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast alpha-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base-regulated serine-threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum- and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in alpha-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2002

Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis.

Ubiquitin functions as a signal for sorting cargo at multiple steps of the endocytic pathway and controls the activity of trans-acting components of the endocytic machinery (reviewed in refs 1, and 2). By contrast to proteasome degradation, which generally requires a polyubiquitin chain that is at least four subunits long, internalization and sorting of endocytic cargo at the late endosome are mediated by mono-ubiquitination. Here, we demonstrate that ubiquitin-interacting motifs (UIMs) found in epsins and Vps27p (ref. 9) from Saccharomyces cerevisiae are required for ubiquitin binding and protein transport. Epsin UIMs are important for the internalization of receptors into vesicles at the plasma membrane. Vps27p UIMs are necessary to sort biosynthetic and endocytic cargo into vesicles that bud into the lumen of a late endosomal compartment, the multivesicular body. We propose that mono-ubiquitin regulates internalization and endosomal sorting by interacting with modular ubiquitin-binding domains in core components of the protein transport machinery. UIM domains are found in a broad spectrum of proteins, consistent with the idea that mono-ubiquitin can function as a regulatory signal to control diverse biological activities.

Adaptor Proteins, Vesicular Transport↗