A Membrane Binding Domain in the Ste5 Scaffold Synergizes with Gβγ Binding to Control Localization and Signaling in Pheromone Response

Winters, Matthew J.; Lamson, Rachel E.; Nakanishi, Hideki; Neiman, Aaron M.; Pryciak, Peter M.

doi:10.1016/j.molcel.2005.08.020

Cited by 103 publications

(179 citation statements)

References 45 publications

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“…For example, a study of all PH domains from the yeast genome showed that PI affinity, while contributing to membrane binding, does not specify subcellular localization (77). As a matter of fact, accumulating evidence shows that the subcellular localization of many peripheral proteins, including those proteins that were known to be recruited to the membrane primarily by protein-protein interactions, is orchestrated by a combination of protein-protein and lipid-protein interactions, and that the relative contribution of two types of interactions varies among proteins (1,78). It should also be noted that an interaction that appears to make a minor contribution in terms of the overall energetics of subcellular targeting of a protein may play a pivotal role in regulating its subcellular localization and activities (1,78).…”

Section: Discussionmentioning

confidence: 99%

Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2α

Stahelin

Karathanassis

Bruzik

et al. 2006

Journal of Biological Chemistry

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Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX domains, we determined the crystal structure of the PX domain from phosphoinositide 3-kinase C2␣ (PI3K-C2␣), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ). To delineate the mechanism by which this PX domain interacts with membranes, we measured the membrane binding of the wild type domain and mutants by surface plasmon resonance and monolayer techniques. This PX domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P 2 binding. The membrane binding of the PX domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P 2 . Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P 2 membrane affinity and specificity when compared with the PI3K-C2␣ C2 domain, demonstrating that high affinity PtdIns(4,5)P 2 binding was facilitated by the PX domain in full-length PI3K-C2␣. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2␣ PX domain interacts with PtdIns(4,5)P 2 -containing membranes and thereby mediates the membrane recruitment of PI3K-C2␣.Numerous cellular processes, including cell signaling and membrane trafficking, involve complex arrays of protein-protein and lipid-protein interactions. Research in the past decade has revealed that a large number of cellular proteins reversibly translocate to specific subcellular locations to form lipid-protein interactions (1, 2). These proteins, collectively known as peripheral proteins, typically contain one or more modular domains specialized in lipid binding (3,4). Common targets of many of these lipid-binding domains are phosphoinositides (PIs), 4 which play a crucial role in cell signaling and membrane trafficking by serving as site-specific membrane signals to modulate the intracellular localization and/or biological activity of effector proteins (5-8). PIs are metabolized by kinases and phosphatases, which catalyze the phosphorylation and dephosphorylation of PI at the 3Ј-, 4Ј-, or 5Ј-position (9 -12). PI 3-kinases (PI3Ks) are enzymes that catalyze the phosphorylation of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), or phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) at the 3Ј-position of the inositol ring (13-15). PI3Ks are divided into three classes (class I, II, and III) based upon their structures and in vitro activities (16,17). Class II PI3K includes PI3K-C2␣, PI3K-C2␤, and PI3K-C2␥. These enzymes phosphorylate PtdIns and PtdIns4P at the 3Ј-position in vitro (18 -21) and act downstream of receptors for growth factors (22), chemokines (23), and integr...

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Section: Discussionmentioning

confidence: 99%

Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2α

Stahelin

Karathanassis

Bruzik

et al. 2006

Journal of Biological Chemistry

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“…and Huntress 1998; Winters et al 2005). Plasma membrane-targeted Ste5 still requires Ste20 p21-activated kinase, the Cdc24 guanine exchange factor and Cdc42 (Pryciak and Huntress 1998;Winters et al 2005).…”

Section: Resultsmentioning

confidence: 99%

“…The Ste5 scaffolding protein recruits the MAP kinases Ste11, Ste7, and Fus3 in response to pheromone to initiate signaling. In turn, Ste5 is recruited to the plasma membrane by pheromone/receptor binding and subsequent interaction with the Gbg dimer Ste4-Ste18, but also interacts with phosphatidyinositol 4,5-bisphosphate [PI(4,5)P 2 ] in the plasma membrane through its pleckstrin homology (PH) domain (Winters et al 2005;Garrenton et al 2006Garrenton et al , 2010. Activation of the pheromone response pathway results in G1 cell cycle arrest, mating-specific gene transcriptional induction, and changes in cytoskeletal structure, which allows for polarized cell growth and alterations in nuclear architecture, ultimately leading to cell fusion and formation of an a/a diploid (Wang and Dohlman 2004).…”

Section: T He Budding Yeast Saccharomyces Cerevisiae Is Viablementioning

confidence: 99%

“…The following plasmids were a gift from Peter Pryciak: 2m HIS3 pGAL1-STE12 (Strickfaden and Pryciak 2008), CEN TRP1 pGAL1-STE11-Cpr (Strickfaden et al 2007), CEN TRP1 pGAL1-GST-Ste11DN (Moskow et al 2000), CEN URA3 STE5-GFPx3 (Strickfaden et al 2007), CEN URA3 pGAL1-STE5 Q59L (Winters et al 2005), and CEN TRP1 pGAL1-STE5DN-CTM (Pryciak and Huntress 1998). The following plasmid was a gift from Jeremy Thorner: CEN LEU2 pGAL1-GST-GFP-PH PLCd (Winters et al 2005;Garrenton et al 2010).…”

Section: Strains Media and Miscellaneous Microbial Techniquesmentioning

confidence: 99%

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The Putative Lipid Transporter, Arv1, Is Required for Activating Pheromone-Induced MAP Kinase Signaling in Saccharomyces cerevisiae

2011

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Saccharomyces cerevisiae haploid cells respond to extrinsic mating signals by forming polarized projections (shmoos), which are necessary for conjugation. We have examined the role of the putative lipid transporter, Arv1, in yeast mating, particularly the conserved Arv1 homology domain (AHD) within Arv1 and its role in this process. Previously it was shown that arv1 cells harbor defects in sphingolipid and glycosylphosphatidylinositol (GPI) biosyntheses and may harbor sterol trafficking defects. Here we demonstrate that arv1 cells are mating defective and cannot form shmoos. They lack the ability to initiate pheromone-induced G1 cell cycle arrest, due to failure to polarize PI(4,5)P 2 and the Ste5 scaffold, which results in weakened MAP kinase signaling activity. A mutant Ste5, Ste5 Q59L, which binds more tightly to the plasma membrane, suppresses the MAP kinase signaling defects of arv1 cells. Filipin staining shows arv1 cells contain altered levels of various sterol microdomains that persist throughout the mating process. Data suggest that the sterol trafficking defects of arv1 affect PI(4,5)P 2 polarization, which causes a mislocalization of Ste5, resulting in defective MAP kinase signaling and the inability to mate. Importantly, our studies show that the AHD of Arv1 is required for mating, pheromone-induced G1 cell cycle arrest, and for sterol trafficking.

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“…Mid1 from Schizosaccharomyces pombe or Ste5 from S. cerevisiae, an NLS or an NLS-like sequence was found within or in close proximity of the helix, which under certain conditions localized the proteins to the nucleus (32,33). (64) is shown in the center of the helix.…”

Section: Termini Of Amino Acid Permeasesmentioning

confidence: 99%

A Plasma Membrane Association Module in Yeast Amino Acid Transporters

Popov‐Čeleketić¹,

Bianchi²,

Ruiz

et al. 2016

Journal of Biological Chemistry

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Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in silico analyses and mutational studies we found that the C-terminal sequences of Gap1, Bap2, Hip1, Tat1, Tat2, Mmp1, Sam3, Agp1, and Gnp1 are about 50 residues long, associate with the PM, and have features that discriminate them from the termini of organellar amino acid transporters. We show that this sequence (named PM asseq ) contains an amphipathic ␣-helix and the FWC signature, which is palmitoylated by palmitoyltransferase Pfa4. Variations of PM asseq , found in different AAPs, lead to different mobilities and localization patterns, whereas the disruption of the sequence has an adverse effect on cell viability. We propose that PM asseq modulates the function and localization of AAPs along the PM. PM asseq is one of the most complex protein signals for plasma membrane association across species and can be used as a delivery vehicle for the PM.Yeast transport amino acids across the plasma membrane (PM), 4 the vacuolar membrane (VM), and to a lesser extent the mitochondrial membrane (1). In the plasma and vacuolar membranes, there are 22 and 11 secondary amino acid transporters, respectively. These transporters are polytopic membrane proteins with 10 -14 transmembrane segments. They belong to three superfamilies: amino acid/polyamine/organocation (APC), major facilitator superfamily (MFS), and amino acid/ polyamine transporter II (AAPTII) (2). The amino acid transporters belonging to the APC superfamily are often referred to as amino acid permeases (AAPs). They are mainly localized in the PM and can be highly specific, e.g. transport only one (enantiomer) amino acid or have a broad range of substrates, like the general amino acid permease Gap1.Alongside the transporter function, additional roles have been proposed for some AAPs. The most prominent example is Gap1, which has a receptor function whereby it signals the protein kinase A (PKA) pathway (3). This so-called transceptor function has also been described for the phosphate transporter Pho84 and the ammonium transporter Mep2 but not for any other AAPs (4, 5). On the other end of the spectrum is Ssy1, an endoplasmic reticulum (ER)-resident AAP member, that plays a role in amino acid sensing but has no transport function (6 -8). The levels of AAPs at the PM are modulated by several transcriptional and post-translational control mechanisms, e.g. nitrogen catabolite repression, general amino acid control, and substrate/stress-induced endocytosis (9 -12). Although for several AAPs many details of these control mechanisms have been elucidated, quite a few questions concerning the spatiotemporal regulation of AAPs remain unanswered. Which mechanism ensures that the function is performed in the right organelle and what determines the positioning of transporters within diff...

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A Membrane Binding Domain in the Ste5 Scaffold Synergizes with Gβγ Binding to Control Localization and Signaling in Pheromone Response

Cited by 103 publications

References 45 publications

Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2α

Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2α

The Putative Lipid Transporter, Arv1, Is Required for Activating Pheromone-Induced MAP Kinase Signaling in Saccharomyces cerevisiae

A Plasma Membrane Association Module in Yeast Amino Acid Transporters

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