Genome-Wide Analysis of Membrane Targeting by S. cerevisiae Pleckstrin Homology Domains

Yu, Jong W.; Mendrola, Jeannine M.; Audhya, Anjon; Singh, Shaneen; Keleti, David; DeWald, Daryll B.; Murray, Diana; Emr, Scott D.; Lemmon, Mark A.

doi:10.1016/s1097-2765(04)00083-8

Cited by 316 publications

(433 citation statements)

References 40 publications

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“…This can certainly not be assumed, however. In our genome-wide analysis of yeast PH domains [27], there are 17 examples (~50% of all S. cerevisiae PH domains) for which we see phosphoinositide binding by dot-blots, but with no supporting evidence from other techniques or from studies of subcellular localization. It remains quite possible that the apparent phosphoinositide binding seen in these cases is simply an artifact of the dot-blot approach.…”

Section: 23mentioning

confidence: 70%

“…For the PLC 1 PH domain, our studies with this SPR approach have given K D estimates of 0.7 ± 0.3 µM for PtdIns(4,5)P 2 binding [27], very similar to our ITC-derived value [39] of 1.66 ± 0.8 µM. Similarly, for PtdIns3P binding by the Hrs1 FYVE domain, this approach gives a K D value of 3.4 ± 0.6 µM, compared with a value of 2.5 µM estimated from the vesicle centrifugation approach outlined above.…”

Section: Discussionmentioning

confidence: 85%

“…It should therefore only be used as a first-pass to determine whether binding to phosphoinositides (or other lipids) can be detected at all. As described in our genome-wide analyses of S. cerevisiae PH and PX domains [26,27], we have often detected phosphoinositide binding using this method that is too weak to be measured by any of the more quantitative approaches outlined later in this article. Other laboratories have reported similar findings [28].…”

Section: Fat Blots/dot-blots/lipid Westerns For Analyzing Binding To mentioning

confidence: 99%

“…First, the state of the phosphoinositides in the spots on the nitrocellulose filter is far from representative of that found in vivo. Second, experience has shown that in many cases phosphoinositide binding that appears robust in dot-blots cannot be detected using any of the other commonly used approaches to study lipid binding [26][27][28]. Conversely, the examples of dynamin's PH domain [31][32][33] and the PH domains from S. cerevisiae proteins Ynl047cp and Yil105cp [27,34] illustrate that phosphoinositide binding that is very weak (and only detectable with dot-blots) can nonetheless be physiologically relevant.…”

Section: 23mentioning

confidence: 99%

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Determining selectivity of phosphoinositide-binding domains

Narayan

Lemmon

2006

Methods

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The burgeoning of phosphoinositide-binding domains and proteins in cellular signaling and trafficking has drawn laboratories from a wide variety of fields into the study of lipid interactions with peripheral membrane proteins. Many different approaches have been developed to assess phosphoinositide binding, some of which are more problematic than others, and some of which can be quantitated more readily than others. With a focus on the methods used in our laboratory, we describe here the considerations that need to be taken into account when establishing -and quantitating -the specific binding of a protein or domain to phosphoinositides in membranes. We also discuss briefly a few examples in which no clear consensus has yet been reached as to the specificity of a given domain or protein because of discrepancies between different commonlyused approaches.

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Section: 23mentioning

confidence: 70%

Section: Discussionmentioning

confidence: 85%

Section: Fat Blots/dot-blots/lipid Westerns For Analyzing Binding To mentioning

confidence: 99%

Section: 23mentioning

confidence: 99%

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Determining selectivity of phosphoinositide-binding domains

Narayan

Lemmon

2006

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“…The CC domain interacts directly with the mitochondrial membrane and is also required for the Num1-dynein interaction [10–12]. The PH domain, which binds with high specificity to PI 4,5 P 2 , targets Num1 to the plasma membrane [13,14], where the protein is found in clusters as well as in a pool that is diffusely localized along the plasma membrane and with cortical ER [3,5,10,15,16]. Cluster formation, which is dependent on the CC domain and an interaction with mitochondria (Figure 1(a)), is critical for the function of Num1 in both mitochondria and dynein anchoring [10,11,17].…”

Section: Introductionmentioning

confidence: 99%

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

et al. 2018

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Organelle distribution is regulated over the course of the cell cycle to ensure that each of the cells produced at the completion of division inherits a full complement of organelles. In yeast, the protein Num1 functions in the positioning and inheritance of two essential organelles, mitochondria and the nucleus. Specifically, Num1 anchors mitochondria as well as dynein to the cell cortex, and this anchoring activity is required for proper mitochondrial distribution and dynein-mediated nuclear inheritance. The assembly of Num1 into clusters at the plasma membrane is critical for both of its anchoring functions. We have previously shown that mitochondria drive the assembly of Num1 clusters and that these mitochondria-assembled Num1 clusters serve as cortical attachment sites for dynein. Here we further examine the role for mitochondria in dynein anchoring. Using a GFP-αGFP nanobody targeting system, we synthetically clustered Num1 on eisosomes to bypass the requirement for mitochondria in Num1 cluster formation. Utilizing this system, we found that mitochondria positively impact the ability of synthetically clustered Num1 to anchor dynein and support dynein function even when mitochondria are no longer required for cluster formation. Thus, the role of mitochondria in regulating dynein function extends beyond simply concentrating Num1; mitochondria likely promote an arrangement of Num1 within a cluster that is competent for dynein anchoring. This functional dependency between mitochondrial and nuclear positioning pathways likely serves as a mechanism to order and integrate major cellular organization systems over the course of the cell cycle.

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Visualization of Cellular Phosphoinositide Pools with GFP‐Fused Protein‐Domains

Balla

Várnai

2009

CP Cell Biology

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This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single‐cell level. The only available technique in live cell applications is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules, when fused to fluorescent proteins, can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular localization, and it has rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. This unit summarizes the design and practical use of these constructs and also reviews important considerations for interpretation of the data obtained by this technique. Curr. Protoc. Cell Biol. 42:24.4.1‐24.4.27. © 2009 by John Wiley & Sons, Inc.

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Genome-Wide Analysis of Membrane Targeting by S. cerevisiae Pleckstrin Homology Domains

Cited by 316 publications

References 40 publications

Determining selectivity of phosphoinositide-binding domains

Determining selectivity of phosphoinositide-binding domains

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

Visualization of Cellular Phosphoinositide Pools with GFP‐Fused Protein‐Domains

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