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1.
Methods Mol Biol ; 2557: 3-15, 2023.
Article in English | MEDLINE | ID: mdl-36512205

ABSTRACT

Fluorescence imaging of live cells allows for the observation of dynamic processes inside cells in real time. Here we describe a strategy to image clathrin-coated vesicle dynamics in a single focal plane at the trans-Golgi network of the yeast Saccharomyces cerevisiae. This method can be readily adapted for live cell imaging of a diverse set of dynamic processes within cells.


Subject(s)
Saccharomyces cerevisiae , trans-Golgi Network , Clathrin-Coated Vesicles , Golgi Apparatus , Clathrin
2.
Free Radic Biol Med ; 154: 105-118, 2020 07.
Article in English | MEDLINE | ID: mdl-32387128

ABSTRACT

Coenzyme Q (CoQ) is an essential player in the respiratory electron transport chain and is the only lipid-soluble antioxidant synthesized endogenously in mammalian and yeast cells. In humans, genetic mutations, pathologies, certain medical treatments, and aging, result in CoQ deficiencies, which are linked to mitochondrial, cardiovascular, and neurodegenerative diseases. The only strategy available for these patients is CoQ supplementation. CoQ supplements benefit a small subset of patients, but the poor solubility of CoQ greatly limits treatment efficacy. Consequently, the efficient delivery of CoQ to the mitochondria and restoration of respiratory function remains a major challenge. A better understanding of CoQ uptake and mitochondrial delivery is crucial to make this molecule a more efficient and effective therapeutic tool. In this study, we investigated the mechanism of CoQ uptake and distribution using the yeast Saccharomyces cerevisiae as a model organism. The addition of exogenous CoQ was tested for the ability to restore growth on non-fermentable medium in several strains that lack CoQ synthesis (coq mutants). Surprisingly, we discovered that the presence of CoQ biosynthetic intermediates impairs assimilation of CoQ into a functional respiratory chain in yeast cells. Moreover, a screen of 40 gene deletions considered to be candidates to prevent exogenous CoQ from rescuing growth of the CoQ-less coq2Δ mutant, identified six novel genes (CDC10, RTS1, RVS161, RVS167, VPS1, and NAT3) as necessary for efficient trafficking of CoQ to mitochondria. The proteins encoded by these genes represent essential steps in the pathways responsible for transport of exogenously supplied CoQ to its functional sites in the cell, and definitively associate CoQ distribution with endocytosis and intracellular vesicular trafficking pathways conserved from yeast to human cells.


Subject(s)
Mitochondrial Diseases , Saccharomyces cerevisiae Proteins , Animals , GTP-Binding Proteins , Humans , Lipids , Microfilament Proteins , N-Terminal Acetyltransferase B , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Ubiquinone/metabolism , Vesicular Transport Proteins
3.
Sci Rep ; 9(1): 4436, 2019 03 14.
Article in English | MEDLINE | ID: mdl-30872642

ABSTRACT

Clathrin coats drive transport vesicle formation from the plasma membrane and in pathways between the trans-Golgi network (TGN) and endosomes. Clathrin adaptors play central roles orchestrating assembly of clathrin coats. The yeast clathrin adaptor-interacting protein Irc6 is an orthologue of human p34, which is mutated in the inherited skin disorder punctate palmoplantar keratoderma type I. Irc6 and p34 bind to clathrin adaptor complexes AP-1 and AP-2 and are members of a conserved family characterized by a two-domain architecture. Irc6 is required for AP-1-dependent transport between the TGN and endosomes in yeast. Here we present evidence that the C-terminal two amino acids of Irc6 are required for AP-1 binding and transport function. Additionally, like the C-terminal domain, the N-terminal domain when overexpressed partially restores AP-1-mediated transport in cells lacking full-length Irc6. These findings support a functional role for Irc6 binding to AP-1. Negative genetic interactions with irc6∆ are enriched for genes related to membrane traffic and nuclear processes, consistent with diverse cellular roles for Irc6.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Cell Membrane/metabolism , Endosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , trans-Golgi Network/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Amino Acid Sequence , Clathrin/metabolism , Golgi Apparatus/metabolism , Mutation , Protein Binding , Protein Domains , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid
4.
Proc Natl Acad Sci U S A ; 114(13): 3433-3438, 2017 03 28.
Article in English | MEDLINE | ID: mdl-28289207

ABSTRACT

Phosphoinositides serve as key membrane determinants for assembly of clathrin coat proteins that drive formation of clathrin-coated vesicles. At the trans-Golgi network (TGN), phosphatidylinositol 4-phosphate (PtdIns4P) plays important roles in recruitment of two major clathrin adaptors, Gga (Golgi-localized, gamma-adaptin ear homology, Arf-binding) proteins and the AP-1 (assembly protein-1) complex. The molecular mechanisms that mediate localization of phosphatidylinositol kinases responsible for synthesis of PtdIns4P at the TGN are not well characterized. We identify two motifs in the yeast phosphatidylinositol 4-kinase, Pik1, which are required for binding to the VHS domain of Gga2. Mutations in these motifs that inhibit Gga2-VHS binding resulted in reduced Pik1 localization and delayed accumulation of PtdIns4P and recruitment of AP-1 to the TGN. The Pik1 homolog in mammals, PI4KIIIß, interacted preferentially with the VHS domain of GGA2 compared with VHS domains of GGA1 and GGA3. Depletion of GGA2, but not GGA1 or GGA3, specifically affected PI4KIIIß localization. These results reveal a conserved role for Gga proteins in regulating phosphatidylinositol 4-kinase function at the TGN.


Subject(s)
1-Phosphatidylinositol 4-Kinase/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , trans-Golgi Network/metabolism , 1-Phosphatidylinositol 4-Kinase/chemistry , 1-Phosphatidylinositol 4-Kinase/genetics , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/genetics , Amino Acid Motifs , Clathrin-Coated Vesicles/metabolism , HeLa Cells , Humans , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Binding , Protein Domains , Protein Transport , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , trans-Golgi Network/genetics
5.
J Cell Biol ; 216(2): 299-301, 2017 02.
Article in English | MEDLINE | ID: mdl-28122956

ABSTRACT

Yeast Vps13 is a member of a conserved protein family that includes human homologues associated with neurodegenerative and developmental disorders. In this issue, De et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201606078) establish direct roles for Vps13 and its surprising binding partner, the calcium-binding centrin Cdc31, in trans-Golgi network (TGN) to endosome traffic and TGN homotypic fusion.


Subject(s)
Calcium-Binding Proteins , trans-Golgi Network , Humans , Protein Transport , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins
6.
Dev Cell ; 37(2): 162-73, 2016 Apr 18.
Article in English | MEDLINE | ID: mdl-27093085

ABSTRACT

Membrane remodeling by BAR (Bin, Amphiphysin, RVS) domain-containing proteins, such as endophilins and amphiphysins, is integral to the process of endocytosis. However, little is known about the regulation of endocytic BAR domain activity. We have identified an interaction between the yeast Rvs167 N-BAR domain and calmodulin. Calmodulin-binding mutants of Rvs167 exhibited defects in endocytic vesicle release. In vitro, calmodulin enhanced membrane tubulation and constriction by wild-type Rvs167 but not calmodulin-binding-defective mutants. A subset of mammalian N-BAR domains bound calmodulin, and co-expression of calmodulin with endophilin A2 potentiated tubulation in vivo. These studies reveal a conserved role for calmodulin in regulating the intrinsic membrane-sculpting activity of endocytic N-BAR domains.


Subject(s)
Calmodulin/metabolism , Cell Membrane/metabolism , Endocytosis/physiology , Liposomes/metabolism , Microfilament Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Animals , Calmodulin/genetics , Constriction , Endocytosis/genetics , Nerve Tissue Proteins/metabolism , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Saccharomyces cerevisiae/genetics
7.
Proc Math Phys Eng Sci ; 471(2182): 20150238, 2015 Oct 08.
Article in English | MEDLINE | ID: mdl-26543397

ABSTRACT

A free-floating wave energy converter (WEC) concept whose power take-off (PTO) system reacts against water inertia is investigated herein. The main focus is the impact of inclining the PTO direction on the system performance. The study is based on a numerical model whose formulation is first derived in detail. Hydrodynamics coefficients are obtained using the linear boundary element method package WAMIT. Verification of the model is provided prior to its use for a PTO parametric study and a multi-objective optimization based on a multi-linear regression method. It is found that inclining the direction of the PTO at around 50° to the vertical is highly beneficial for the WEC performance in that it provides a high capture width ratio over a broad region of the wave period range.

8.
Front Biosci (Landmark Ed) ; 18(3): 862-91, 2013 06 01.
Article in English | MEDLINE | ID: mdl-23747853

ABSTRACT

Since the identification of clathrin as a vesicular coat protein, numerous studies have contributed to our understanding of the role of clathrin and clathrin-mediated trafficking pathways in cell function. The budding yeast, Saccharomyces cerevisiae, offers a wealth of highly developed approaches that have been applied to study clathrin-mediated trafficking events, most of which are conserved in mammalian cells. Here we review the function of clathrin and clathrin adaptors in yeast. We also discuss the role of these proteins in human disease and how certain pathogens have co-opted trafficking pathways for their own use. These studies highlight the advantages of studying complex trafficking events using yeast as a model.


Subject(s)
Clathrin/metabolism , Disease , Saccharomyces cerevisiae/metabolism , Endocytosis , Endosomes/metabolism , Humans , trans-Golgi Network/metabolism
9.
Mol Biol Cell ; 23(22): 4416-29, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22993212

ABSTRACT

Clathrin coat accessory proteins play key roles in transport mediated by clathrin-coated vesicles. Yeast Irc6p and the related mammalian p34 are putative clathrin accessory proteins that interact with clathrin adaptor complexes. We present evidence that Irc6p functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p. The crystal structure of the Irc6p N-terminal domain revealed a G-protein fold most related to small G proteins of the Rab and Arf families. However, Irc6p lacks G-protein signature motifs and high-affinity GTP binding. Also, mutant Irc6p lacking candidate GTP-binding residues retained function. Mammalian p34 rescued growth defects in irc6 cells, indicating functional conservation, and modeling predicted a similar N-terminal fold in p34. Irc6p and p34 also contain functionally conserved C-terminal regions. Irc6p/p34-related proteins with the same two-part architecture are encoded in genomes of species as diverse as plants and humans. Together these results define Irc6p/p34 as a novel type of conserved clathrin accessory protein and founding members of a new G protein-like family.


Subject(s)
Adaptor Proteins, Vesicular Transport/physiology , Monomeric GTP-Binding Proteins/physiology , Saccharomyces cerevisiae Proteins/physiology , ADP-Ribosylation Factor 1/chemistry , Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/metabolism , Amino Acid Sequence , Biological Transport , Clathrin/metabolism , Conserved Sequence , Crystallography, X-Ray , Endosomes/metabolism , Molecular Sequence Data , Monomeric GTP-Binding Proteins/chemistry , Monomeric GTP-Binding Proteins/metabolism , Protein Interaction Mapping , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , rab GTP-Binding Proteins/chemistry , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , trans-Golgi Network/metabolism , trans-Golgi Network/physiology
10.
J Biol Chem ; 287(21): 17398-17407, 2012 May 18.
Article in English | MEDLINE | ID: mdl-22457357

ABSTRACT

Membrane traffic is an essential process that allows protein and lipid exchange between the endocytic, lysosomal, and secretory compartments. Clathrin-mediated traffic between the trans-Golgi network and endosomes mediates responses to the environment through the sorting of biosynthetic and endocytic protein cargo. Traffic through this pathway is initiated by the controlled assembly of a clathrin-adaptor protein coat on the cytosolic surface of the originating organelle. In this process, clathrin is recruited by different adaptor proteins that act as a bridge between clathrin and the transmembrane cargo proteins to be transported. Interactions between adaptors and clathrin and between different types of adaptors lead to the formation of a densely packed protein network within the coat. A key unresolved issue is how the highly complex adaptor-clathrin interaction and adaptor-adaptor interaction landscape lead to the correct spatiotemporal assembly of the clathrin coat. Here we report the discovery of a new autoregulatory motif within the clathrin adaptor Gga2 that drives synergistic binding of Gga2 to clathrin and the adaptor Ent5. This autoregulation influences the temporal and/or spatial location of the Gga2-Ent5 interaction. We propose that this synergistic binding provides built-in regulation to ensure the correct assembly of clathrin coats.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Clathrin/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , trans-Golgi Network/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Amino Acid Motifs , Biological Transport, Active/physiology , Clathrin/genetics , Endosomes/genetics , Endosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , trans-Golgi Network/genetics
11.
Nat Cell Biol ; 14(3): 239-48, 2012 Feb 19.
Article in English | MEDLINE | ID: mdl-22344030

ABSTRACT

Clathrin-coated vesicles mediate endocytosis and transport between the trans-Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo and membranes. Two main types of clathrin adaptor act in TGN-endosome traffic: GGA proteins and the AP-1 complex. Here we characterize the relationship between GGA proteins, AP-1 and other TGN clathrin adaptors using live-cell and super-resolution microscopy in yeast. We present evidence that GGA proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PtdIns(4)P) levels at the TGN slow or uncouple AP-1 coat assembly from GGA coat assembly. Conversely, enhanced PtdIns(4)P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PtdIns(4)-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PtdIns(4)P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN.


Subject(s)
1-Phosphatidylinositol 4-Kinase/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Phosphatidylinositol Phosphates/metabolism , Saccharomyces cerevisiae Proteins/metabolism , trans-Golgi Network/metabolism , 1-Phosphatidylinositol 4-Kinase/genetics , Adaptor Protein Complex 1/genetics , Adaptor Protein Complex 1/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Clathrin/metabolism , Clathrin-Coated Vesicles/metabolism , Endosomes/metabolism , Immunoblotting , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Mutation , Protein Binding , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Time-Lapse Imaging
12.
Dev Cell ; 18(5): 687-8, 2010 May 18.
Article in English | MEDLINE | ID: mdl-20493800

ABSTRACT

Dynamin family members are large GTPases that assemble into multimeric spirals. These spirals promote membrane fission or fusion, or they inhibit processes such as viral replication. Two new studies by Chappie et al. and Gao et al. in a recent issue of Nature identify interactions between subunits of dynamin spirals, advancing mechanistic understanding of dynamin function.

13.
EMBO J ; 29(6): 1033-44, 2010 Mar 17.
Article in English | MEDLINE | ID: mdl-20150898

ABSTRACT

During clathrin-mediated endocytosis, adaptor proteins play central roles in coordinating the assembly of clathrin coats and cargo selection. Here we characterize the binding of the yeast endocytic adaptor Sla1p to clathrin through a variant clathrin-binding motif that is negatively regulated by the Sla1p SHD2 domain. The crystal structure of SHD2 identifies the domain as a sterile alpha-motif (SAM) domain and shows a propensity to oligomerize. By co-immunoprecipitation, Sla1p binds to clathrin and self-associates in vivo. Mutations in the clathrin-binding motif that abolish clathrin binding and structure-based mutations in SHD2 that impede self-association result in endocytosis defects and altered dynamics of Sla1p assembly at the sites of endocytosis. These results define a novel mechanism for negative regulation of clathrin binding by an adaptor and suggest a role for SAM domains in clathrin-mediated endocytosis.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Clathrin/metabolism , Endocytosis/physiology , Adaptor Proteins, Vesicular Transport/chemistry , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Clathrin/chemistry , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Molecular Sequence Data , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism
14.
Mol Biol Cell ; 20(5): 1592-604, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19116312

ABSTRACT

The evolutionarily conserved adaptor protein-3 (AP-3) complex mediates cargo-selective transport to lysosomes and lysosome-related organelles. To identify proteins that function in AP-3-mediated transport, we performed a genome-wide screen in Saccharomyces cerevisiae for defects in the vacuolar maturation of alkaline phosphatase (ALP), a cargo of the AP-3 pathway. Forty-nine gene deletion strains were identified that accumulated precursor ALP, many with established defects in vacuolar protein transport. Maturation of a vacuolar membrane protein delivered via a separate, clathrin-dependent pathway, was affected in all strains except those with deletions of YCK3, encoding a vacuolar type I casein kinase; SVP26, encoding an endoplasmic reticulum (ER) export receptor for ALP; and AP-3 subunit genes. Subcellular fractionation and fluorescence microscopy revealed ALP transport defects in yck3Delta cells. Characterization of svp26Delta cells revealed a role for Svp26p in ER export of only a subset of type II membrane proteins. Finally, ALP maturation kinetics in vac8Delta and vac17Delta cells suggests that vacuole inheritance is important for rapid generation of proteolytically active vacuolar compartments in daughter cells. We propose that the cargo-selective nature of the AP-3 pathway in yeast is achieved by AP-3 and Yck3p functioning in concert with machinery shared by other vacuolar transport pathways.


Subject(s)
Adaptor Protein Complex 3/physiology , Alkaline Phosphatase/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Adaptor Protein Complex 3/genetics , Alkaline Phosphatase/analysis , Casein Kinase I/genetics , Casein Kinase I/metabolism , Casein Kinase I/physiology , Gene Deletion , Genome, Fungal , Green Fluorescent Proteins/analysis , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/physiology , Protein Subunits/genetics , Protein Transport/genetics , Protein Transport/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins
15.
Methods Mol Biol ; 457: 13-27, 2008.
Article in English | MEDLINE | ID: mdl-19066016

ABSTRACT

A variety of Saccharomyces cerevisiae strain libraries allow for systematic analysis of strains bearing gene deletions, repressible genes, overexpressed genes, or modified genes on a genome-wide scale. Here we introduce a method for culturing yeast strains in 96-well format to achieve log-phase growth and a high-throughput technique for generating whole-cell protein extracts from these cultures using sodium dodecyl sulfate and heat lysis. We subsequently describe a procedure to analyze these whole-cell extracts by immunoblotting for alkaline phosphatase and carboxypeptidase yscS to identify strains with defects in protein transport pathways or protein glycosylation. These methods should be readily adaptable to many different areas of interest.


Subject(s)
Biochemistry/methods , Immunoblotting/methods , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae/metabolism , Alkaline Phosphatase/metabolism , Carboxypeptidases/metabolism , Electrophoresis, Polyacrylamide Gel , Fermentation , Genes, Fungal , Glycosylation , Protein Transport , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development
16.
Proc Natl Acad Sci U S A ; 104(15): 6235-40, 2007 Apr 10.
Article in English | MEDLINE | ID: mdl-17404221

ABSTRACT

Small molecule inhibitors provide powerful tools to characterize highly dynamic and complex eukaryotic cell pathways such as those mediating membrane traffic. However, a lack of easy and generalizable assays has constrained identification of novel inhibitors despite availability of diverse chemical libraries. Here, we report a facile growth-based strategy in yeast to screen for pathway-specific inhibitors. The approach uses well characterized synthetic genetic growth defects to guide design of cells genetically sensitized for inhibition of chosen pathways. With this strategy, we identified a family of piperazinyl phenylethanone compounds as inhibitors of traffic between the trans-Golgi network (TGN) and endosomes that depends on the clathrin adaptor complex AP-1. The compounds did not significantly alter other trafficking pathways involving the TGN or endosomes, indicating specificity. Compound treatment also altered localization of AP-1 in mammalian cells. These previously uncharacterized inhibitors will be useful for future studies of clathrin-mediated transport in yeast, and potentially in other organisms. Furthermore, the easily automated technology should be adaptable for identification of inhibitors of other cellular processes.


Subject(s)
Adaptor Protein Complex 1/antagonists & inhibitors , Adaptor Proteins, Vesicular Transport/genetics , Endosomes/metabolism , Organic Chemicals/metabolism , Saccharomyces cerevisiae Proteins/genetics , trans-Golgi Network/metabolism , Adaptor Protein Complex 1/metabolism , Cell Survival , Chitin/metabolism , Genes, Lethal/genetics , Molecular Structure , Mutation/genetics , Protein Transport/physiology , Yeasts
17.
EMBO J ; 26(7): 1963-71, 2007 Apr 04.
Article in English | MEDLINE | ID: mdl-17363896

ABSTRACT

Adaptor proteins play important endocytic roles including recognition of internalization signals in transmembrane cargo. Sla1p serves as the adaptor for uptake of transmembrane proteins containing the NPFxD internalization signal, and is essential for normal functioning of the actin cytoskeleton during endocytosis. The Sla1p homology domain 1 (SHD1) within Sla1p is responsible for recognition of the NPFxD signal. This study presents the NMR structure of the NPFxD-bound state of SHD1 and a model for the protein-ligand complex. The alpha+beta structure of the protein reveals an SH3-like topology with a solvent-exposed hydrophobic ligand binding site. NMR chemical shift perturbations and effects of structure-based mutations on ligand binding in vitro define residues that are key for NPFxD binding. Mutations that abolish ligand recognition in vitro also abolish NPFxD-mediated receptor internalization in vivo. Thus, SHD1 is a novel functional domain based on SH3-like topology, which employs a unique binding site to recognize the NPFxD endocytic internalization signal. Its distant relationship with the SH3 fold endows this superfamily with a new role in endocytosis.


Subject(s)
Amino Acid Motifs , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Endocytosis , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Binding Sites , Cell Wall/metabolism , Cytoskeletal Proteins , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Binding , Protein Folding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Solutions , Substrate Specificity
18.
Mol Biol Cell ; 18(1): 57-65, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17065552

ABSTRACT

The actin-associated protein Sla1p, through its SHD1 domain, acts as an adaptor for the NPFX(1,2)D endocytic targeting signal in yeast. Here we report that Wsc1p, a cell wall stress sensor, depends on this signal-adaptor pair for endocytosis. Mutation of NPFDD in Wsc1p or expression of Sla1p lacking SHD1 blocked Wsc1p internalization. By live cell imaging, endocytically defective Wsc1p was not concentrated at sites of endocytosis. Polarized distribution of Wsc1p to regions of cell growth was lost in the absence of endocytosis. Mutations in genes necessary for endosome to Golgi traffic caused redistribution of Wsc1p from the cell surface to internal compartments, indicative of recycling. Inhibition of Wsc1p endocytosis caused defects in polarized deposition of the cell wall and increased sensitivity to perturbation of cell wall synthesis. Our results reveal that the NPFX(1,2)D-Sla1p system is responsible for directing Wsc1p into an endocytosis and recycling pathway necessary to maintain yeast cell wall polarity. The dynamic localization of Wsc1p, a sensor of the extracellular wall in yeast, resembles polarized distribution of certain extracellular matrix-sensing integrins through endocytic recycling.


Subject(s)
Cell Polarity , Cell Wall/metabolism , Endocytosis , Membrane Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Signal Transduction , Amino Acid Motifs , Carrier Proteins/metabolism , Cell Membrane/metabolism , Cell Wall/ultrastructure , Cytoskeletal Proteins , Membrane Proteins/chemistry , Protein Binding , Protein Structure, Tertiary , Protein Transport , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry
19.
J Biol Chem ; 281(46): 35520-30, 2006 Nov 17.
Article in English | MEDLINE | ID: mdl-16973627

ABSTRACT

The AGCVIIIa kinases of Arabidopsis are members of the eukaryotic PKA, PKG, and PKC group of regulatory kinases. One AGCVIIIa kinase, PINOID (PID), plays a fundamental role in the asymmetrical localization of membrane proteins during polar auxin transport. The remaining 16 AGCVIIIa genes have not been associated with single mutant phenotypes, suggesting that the corresponding kinases function redundantly. Consistent with this idea, we find that the genes encoding the Arabidopsis AGCVIIIa kinases have spatially distinct, but overlapping, expression domains. Here we show that the majority of Arabidopsis AGCVIIIa kinases are substrates for the 3-phosphoinositide-dependent kinase 1 (PDK1) and that trans-phosphorylation by PDK1 correlates with activation of substrate AGCVIIIa kinases. Mutational analysis of two conserved regulatory domains was used to demonstrate that sequences located outside of the C-terminal PDK1 interaction (PIF) domain and the activation loop are required for functional interactions between PDK1 and its substrates. A subset of GFP-tagged AGCVIIIa kinases expressed in Saccharomyces cerevisiae and tobacco BY-2 cells were preferentially localized to the cytoplasm (AGC1-7), nucleus (WAG1 and KIPK), and the cell periphery (PID). We present evidence that PID insertion domain sequences are sufficient to direct the observed peripheral localization. We find that PID specifically but non-selectively binds to phosphoinositides and phosphatidic acid, suggesting that PID might directly interact with the plasma membrane through protein-lipid interactions. The initial characterization of the AGCVIIIa kinases presented here provides a framework for elucidating the physiological roles of these kinases in planta.


Subject(s)
Arabidopsis/enzymology , Protein Kinases/metabolism , 3-Phosphoinositide-Dependent Protein Kinases , Amino Acid Sequence , Enzyme Activation , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Molecular Sequence Data , Protein Kinases/genetics , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Protein Transport
20.
Cell ; 126(3): 611-25, 2006 Aug 11.
Article in English | MEDLINE | ID: mdl-16901791

ABSTRACT

Discovering target and off-target effects of specific compounds is critical to drug discovery and development. We generated a compendium of "chemical-genetic interaction" profiles by testing the collection of viable yeast haploid deletion mutants for hypersensitivity to 82 compounds and natural product extracts. To cluster compounds with a similar mode-of-action and to reveal insights into the cellular pathways and proteins affected, we applied both a hierarchical clustering and a factorgram method, which allows a gene or compound to be associated with more than one group. In particular, tamoxifen, a breast cancer therapeutic, was found to disrupt calcium homeostasis and phosphatidylserine (PS) was recognized as a target for papuamide B, a cytotoxic lipopeptide with anti-HIV activity. Further, the profile of crude extracts resembled that of its constituent purified natural product, enabling detailed classification of extract activity prior to purification. This compendium should serve as a valuable key for interpreting cellular effects of novel compounds with similar activities.


Subject(s)
Drug Evaluation, Preclinical/methods , Drug Resistance/genetics , Gene Expression Profiling/methods , Pharmaceutical Preparations/metabolism , Yeasts/drug effects , Yeasts/genetics , Antineoplastic Agents, Hormonal/pharmacology , Antiviral Agents/pharmacology , Cluster Analysis , Depsipeptides/pharmacology , Fungal Proteins/drug effects , Fungal Proteins/genetics , Fungal Proteins/metabolism , Molecular Structure , Mutation/drug effects , Mutation/genetics , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/classification , Phosphatidylserines/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Tamoxifen/pharmacology , Yeasts/metabolism
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