Volume 13, Issue 5, Pages (March 2004)

Slides:



Advertisements
Similar presentations
Volume 143, Issue 6, Pages (December 2010)
Advertisements

Yeast Tgl3p expressed in HeLa cells localizes to lipid droplets.
The vacuolar-ATPase B1 subunit in distal tubular acidosis: novel mutations and mechanisms for dysfunction  D.G. Fuster, J. Zhang, X.-S. Xie, O.W. Moe 
Volume 23, Issue 6, Pages (May 2018)
Volume 143, Issue 6, Pages (December 2010)
B.Alexander Yi, Yu-Fung Lin, Yuh Nung Jan, Lily Yeh Jan  Neuron 
Volume 26, Issue 1, Pages (April 2007)
Volume 23, Issue 10, Pages (October 2015)
Spindle Position Is Coordinated with Cell-Cycle Progression through Establishment of Mitotic Exit-Activating and -Inhibitory Zones  Leon Y. Chan, Angelika.
The Structure of the Cytoplasmic Domain of the Chloride Channel ClC-Ka Reveals a Conserved Interaction Interface  Sandra Markovic, Raimund Dutzler  Structure 
Volume 20, Issue 7, Pages (April 2010)
Volume 7, Issue 2, Pages (February 2001)
The Arl4 Family of Small G Proteins Can Recruit the Cytohesin Arf6 Exchange Factors to the Plasma Membrane  Irmgard Hofmann, Amanda Thompson, Christopher M.
Volume 23, Issue 6, Pages (December 2012)
Sherilyn Grill, Valerie M. Tesmer, Jayakrishnan Nandakumar 
The Unstructured C-Terminal Tail of the Clamp Subunit Ddc1 Activates Mec1/ATR via Two Distinct Mechanisms  Vasundhara M. Navadgi-Patil, Peter M.
Volume 45, Issue 5, Pages (March 2012)
Volume 15, Issue 9, Pages (September 2008)
Crystal Structure of the Rab9A-RUTBC2 RBD Complex Reveals the Molecular Basis for the Binding Specificity of Rab9A with RUTBC2  Zhe Zhang, Shanshan Wang,
Volume 23, Issue 11, Pages (November 2015)
Volume 9, Issue 4, Pages (April 2002)
Direct Observation of Single MuB Polymers
Volume 19, Issue 2, Pages (February 2011)
Diabetes Mutations Delineate an Atypical POU Domain in HNF-1α
Volume 1, Issue 2, Pages (April 2007)
Volume 11, Issue 5, Pages (May 2003)
Volume 11, Issue 1, Pages (July 1999)
Volume 20, Issue 3, Pages (March 2004)
Volume 18, Issue 10, Pages (May 2008)
Volume 17, Issue 1, Pages (January 2005)
Stt4 PI 4-Kinase Localizes to the Plasma Membrane and Functions in the Pkc1- Mediated MAP Kinase Cascade  Anjon Audhya, Scott D. Emr  Developmental Cell 
Andrew H. Huber, W.James Nelson, William I. Weis  Cell 
Targeting of Golgi-Specific Pleckstrin Homology Domains Involves Both PtdIns 4- Kinase-Dependent and -Independent Components  Timothy P. Levine, Sean Munro 
Specificity Determinants in Phosphoinositide Dephosphorylation
Hyunsuk Suh, Dane Z. Hazelbaker, Luis M. Soares, Stephen Buratowski 
Volume 16, Issue 3, Pages (March 2008)
Volume 5, Issue 3, Pages (May 2012)
Structure, Exchange Determinants, and Family-Wide Rab Specificity of the Tandem Helical Bundle and Vps9 Domains of Rabex-5  Anna Delprato, Eric Merithew,
Volume 13, Issue 2, Pages (February 2005)
Volume 23, Issue 6, Pages (May 2018)
Volume 7, Issue 4, Pages (April 2005)
Structural Basis for Specific Recognition of Reelin by Its Receptors
Volume 7, Issue 3, Pages (March 2008)
Microtubule Interaction Site of the Kinesin Motor
Christopher G Burd, Scott D Emr  Molecular Cell 
Rong Guan, Dai Han, Stephen C. Harrison, Tomas Kirchhausen  Structure 
Volume 11, Issue 2, Pages (February 2003)
A Computational Model for the Electrostatic Sequestration of PI(4,5)P2 by Membrane- Adsorbed Basic Peptides  Jiyao Wang, Alok Gambhir, Stuart McLaughlin,
Volume 26, Issue 1, Pages (April 2007)
Volume 6, Issue 4, Pages (October 2000)
Satoru Funamoto, Ruedi Meili, Susan Lee, Lisa Parry, Richard A. Firtel 
Volume 29, Issue 6, Pages (March 2008)
Volume 85, Issue 5, Pages (May 1996)
Volume 14, Issue 2, Pages (August 2008)
Volume 46, Issue 2, Pages (April 2012)
Structure of the Staphylococcus aureus AgrA LytTR Domain Bound to DNA Reveals a Beta Fold with an Unusual Mode of Binding  David J. Sidote, Christopher.
Volume 30, Issue 5, Pages (June 2008)
Protein Structure Prediction: Inroads to Biology
Crystal Structure of a Polymeric Immunoglobulin Binding Fragment of the Human Polymeric Immunoglobulin Receptor  Agnes E. Hamburger, Anthony P. West,
Volume 19, Issue 8, Pages (August 2011)
Volume 30, Issue 3, Pages (May 2008)
Volume 11, Issue 2, Pages (February 2003)
Electrostatic Control of the Membrane Targeting of C2 Domains
The Structure of the GGA1-GAT Domain Reveals the Molecular Basis for ARF Binding and Membrane Association of GGAs  Brett M. Collins, Peter J. Watson,
Parul Mishra, Julia M. Flynn, Tyler N. Starr, Daniel N.A. Bolon 
Pleckstrin Homology Domains: Two Halves Make a Hole?
Volume 28, Issue 1, Pages (January 2014)
Volume 105, Issue 2, Pages (July 2013)
Volume 15, Issue 5, Pages (May 2007)
Volume 95, Issue 2, Pages (October 1998)
Presentation transcript:

Volume 13, Issue 5, Pages 677-688 (March 2004) Genome-Wide Analysis of Membrane Targeting by S. cerevisiae Pleckstrin Homology Domains  Jong W. Yu, Jeannine M. Mendrola, Anjon Audhya, Shaneen Singh, David Keleti, Daryll B. DeWald, Diana Murray, Scott D. Emr, Mark A. Lemmon  Molecular Cell  Volume 13, Issue 5, Pages 677-688 (March 2004) DOI: 10.1016/S1097-2765(04)00083-8 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 Phosphoinositide Binding Specificity of Yeast PH Domains Using a Lipid Overlay Method In the upper panel, a representative lipid overlay experiment is shown for each specificity group (except the “no binding” group). In the lower panel, a semiquantitative representation of lipid overlay results for each PH domain is shown. The phosphoinositide that bound most strongly to each PH domain was arbitrarily scored as 100%. For a representative experiment, binding to other phosphoinositides is expressed as a percentage of this value and the boxes colored according to the legend. This scoring scheme does not allow comparison of affinities between PH domains, but color variation gives an impression of specificity. Molecular Cell 2004 13, 677-688DOI: (10.1016/S1097-2765(04)00083-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 Surface Plasmon Resonance Analysis of Phosphoinositide Binding by Yeast PH Domains (A) Each GST/PH fusion was flowed at ≥3 μM over a sensorchip containing 3% (mole/mole) PtdIns(4,5)P2 in DOPC. PH domains that gave signals >1000 RUs are listed in bold type and were further analyzed. The PH domains from Bem2p, Bud4p, Sip3p, Ybl060wp, Yhr131cp, and Ynl144cp could not be produced in sufficient quantities for analysis with this approach. (B) PH domains that bound significantly in (A) were subjected to more detailed binding analysis. Monomeric PH domains were flowed at a series of concentrations across surfaces containing 3% (mole/mole) PtdIns(4,5)P2, PtdIns(3,5)P2, PtdIns(3)P, or PtdIns(4)P. Binding signals are plotted against protein concentration, and the best-fit curves (with KD values reported in Table 1) are superimposed. Data for PLCδ-PH are shown for comparison. Results are representative of at least three repeats, with errors given in Table 1. (C) Phosphoinositide binding curves for the GST/Num1p-PH protein, as described in (B). Data for GST/PLCδ-PH are shown for comparison. Molecular Cell 2004 13, 677-688DOI: (10.1016/S1097-2765(04)00083-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 Subcellular Targeting of EGFP-Fused Yeast PH Domains in HeLa and Yeast Cells Fluorescence micrographs are shown only for cases in which significant targeting was observed. Results for all other PH domains in yeast are given in Table 2. Molecular Cell 2004 13, 677-688DOI: (10.1016/S1097-2765(04)00083-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 Membrane Targeting of Wild-Type and Mutated Yeast PH Domains Assessed by Ras Rescue (A) Serial dilutions of cdc25ts yeast cultures expressing the noted PH domain/RasQ61L(Δf) fusion were spotted onto duplicate selection plates lacking leucine and incubated at the permissive temperature (25°C) or restrictive temperature (37°C) for 7 to 8 days. Membrane targeting by the fused PH domain is required for yeast growth at 37°C (Isakoff et al., 1998). At the right of each experiment it is noted whether the EGFP/PH fusion was localized in yeast cells to the plasma membrane (PM), Golgi (G), nucleus (Nuc), or only the cytoplasm (−). (B) Sequence alignment of the β1/β2 loop region of yeast PH domains that were membrane targeted as EGFP fusion proteins. The basic residues colored red [critical for PtdIns(4,5)P2 binding in the case of PLCδ-PH] were simultaneously mutated to alanine to disrupt each predicted phosphoinositide binding site. (C) Ras rescue experiments show that mutation of the presumed phosphoinositide binding site abolishes the ability of all eight PH domains to drive membrane recruitment. Western blot controls (data not shown) confirmed that Ras/PH fusion expression was not impaired by these mutations. Molecular Cell 2004 13, 677-688DOI: (10.1016/S1097-2765(04)00083-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 5 EGFP-PH Targeting in Yeast Cells with Altered PtdIns(4,5)P2 or PtdIns(4)P Levels Localization of the eight noted PH domains (fused to EGFP) was analyzed in mss4ts cells [with reduced PtdIns(4,5)P2 levels], grown to mid-log phase, and then incubated for 45 min at either 26°C or 37°C [where PtdIns(4,5)P2 levels are further reduced], before being examined live by fluorescence microscopy. Localization of the same EGFP fusions in sjl1Δ cells [with elevated PM PtdIns(4,5)P2] and sac1Δ cells [with elevated PM PtdIns(4)P] is also shown. Molecular Cell 2004 13, 677-688DOI: (10.1016/S1097-2765(04)00083-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 6 Electrostatic Characteristics of Modeled Yeast PH Domains GRASP (Nicholls et al., 1991) representations are shown for the 19 PH domains for which reliable models could be obtained. Well-modeled PH domains were subdivided into three groups: those with strong positive potential arising from the β1/β2 loop (A), those with positive potential arising from the β1/β2 and β5/β6 loops (B), and those with little or no positive potential (C). Each PH domain model is shown in the same orientation, with the phosphoinositide binding side at the bottom of the panel. Alpha carbon traces are presented as white worms, and equipotential profiles are represented as blue (+25 mV) and red (−25 mV) meshes. Molecular Cell 2004 13, 677-688DOI: (10.1016/S1097-2765(04)00083-8) Copyright © 2004 Cell Press Terms and Conditions