Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins

Phosphoinositides (PtdInsPs) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological treatments dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized to EEA1-positive endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and tandem FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

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The effector TepP mediates the recruitment and activation of Phosphoinositide 3 Kinase on early Chlamydia trachomatis vacuoles

10.1101/132282 ◽

2017 ◽

Author(s):

Victoria Carpenter ◽

Yi-Shan Chen ◽

Lee Dolat ◽

Raphael H. Valdivia

Keyword(s):

Chlamydia Trachomatis ◽

Epithelial Cells ◽

Membrane Trafficking ◽

Adaptor Protein ◽

Membrane Dynamics ◽

Effector Proteins ◽

Type I ◽

Type I Ifn ◽

Bacterial Replication ◽

Phosphoinositide 3 Kinase

ABSTRACTChlamydia trachomatis delivers multiple Type 3 secreted effector proteins to host epithelial cells to manipulate cytoskeletal functions, membrane dynamics and signaling pathways. TepP is the most abundant effector protein secreted early in infection but its molecular function is poorly understood. In this report, we provide evidence that TepP is important for bacterial replication in cervical epithelial cells, the activation of Type I IFN genes, and the recruitment of Class I phosphoinositide 3 kinases (PI3K) and the signaling adaptor protein CrkL to nascent pathogen-containing vacuoles (inclusions). We also show that TepP is a target of tyrosine phosphorylation by Src kinases but these modifications do not appear to influence the recruitment of PI3K or CrkL. The translocation of TepP correlated with an increase in the intracellular pools of phosphoinositide 3,4,5 triphosphate but not the activation of the pro-survival kinase Akt, suggesting that TepP-mediated activation of PI3K is spatially restricted to early inclusions. Furthermore, we linked PI3K activity to the dampening of transcription of Type I IFN induced genes early in infection. Overall, these findings indicate that TepP can modulate cell signaling and potentially membrane trafficking events by spatially restricted activation of PI3K.

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The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells

10.32920/ryerson.14661819 ◽

2021 ◽

Author(s):

Krishna Chintaluri

Keyword(s):

Toxoplasma Gondii ◽

Membrane Trafficking ◽

Mammalian Cells ◽

Effector Proteins ◽

Ph Domain ◽

Fyve Domain ◽

Px Domain ◽

Early Endosomes ◽

Phosphatidylinositol 3

Phosphoinositides (PtdInsPs) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological treatments dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized to EEA1-positive endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and tandem FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

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FYVE-finger proteins--effectors of an inositol lipid

Journal of Cell Science ◽

10.1242/jcs.112.23.4175 ◽

1999 ◽

Vol 112 (23) ◽

pp. 4175-4183 ◽

Cited By ~ 10

Author(s):

H. Stenmark ◽

R. Aasland

Keyword(s):

Membrane Trafficking ◽

Current Knowledge ◽

Structural Basis ◽

Membrane Recruitment ◽

Cellular Processes ◽

Receptor Signalling ◽

Cytoskeletal Function ◽

Phosphoinositide 3 Kinase ◽

Derivatives Of ◽

Review Current

The binding of cytosolic proteins to specific intracellular membranes containing phosphorylated derivatives of phosphatidylinositol (PtdIns) is a common theme in vital cellular processes, such as cytoskeletal function, receptor signalling and membrane trafficking. Recently, several potential effectors of the phosphoinositide 3-kinase product PtdIns 3-phosphate (PtdIns(3)P) have emerged through the observation that a conserved zinc-finger-like domain, the FYVE-finger, binds specifically to this lipid. Here we review current knowledge about the structural basis for the FYVE-PtdIns(3)P interaction, its role in membrane recruitment of proteins and the functions of FYVE-finger proteins in membrane trafficking and other cellular processes.

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Establishing a model to demonstrate physical and mathematical properties of chromatin fibres in fission yeast cells - Research in the Molecular Biophysics Lab at Hiroshima University

Impact ◽

10.21820/23987073.2018.3.89 ◽

2018 ◽

Vol 2018 (3) ◽

pp. 89-91

Author(s):

Shin-ichi Tate

Keyword(s):

Molecular Biology ◽

Yeast Cells ◽

Molecular Architecture ◽

Ministry Of Education ◽

Cellular Functions ◽

Sports Science ◽

Cellular Events ◽

And Function ◽

Education Culture ◽

Open The Doors

The field of molecular biology has provided great insights into the structure and function of key molecules. Thanks to this area of research, we can now grasp the biological details of DNA and have characterised an enormous number of molecules in massive data bases. These 'biological periodic tables' have allowed scientists to connect molecules to particular cellular events, furthering scientific understanding of biological processes. However, molecular biology has yet to answer questions regarding 'higher-order' molecular architecture, such as that of chromatin. Chromatin is the molecular material that serves as the building block for chromosomes, the structures that carry an organism's genetic information inside of the cell's nucleus. Understanding the physical properties of chromatin is crucial in developing a more thorough picture of how chromatin's structure relate to its key cellular functions. Moreover, by establishing a physical model of chromatin, scientists will be able to open the doors into the true inner workings of the cell nucleus. Professor Shin-ichi Tate and his team of researchers at Hiroshima University's Research Center for the Mathematics on Chromatin Live Dynamics (RcMcD), are attempting to do just that. Through a five-year grant funded by the Platform for Dynamic Approaches to Living Systems from the Ministry of Education, Culture, Sports, Science and Technology, Tate is aiming to gain a clearer understanding of the structure and dynamics of chromatin.

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An FYVE-Domain-Containing Protein, PsFP1, Is Involved in Vegetative Growth, Oxidative Stress Response and Virulence of Phytophthora sojae

International Journal of Molecular Sciences ◽

10.3390/ijms22126601 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6601

Author(s):

Jinhui Zhang ◽

Xiaoran Du ◽

Xin Zhou ◽

Duo Jin ◽

Jianqiang Miao ◽

...

Keyword(s):

Oxidative Stress ◽

Membrane Trafficking ◽

Vegetative Growth ◽

Genome Mining ◽

Gene Replacement ◽

Wide Distribution ◽

Stress Sensitivity ◽

Phytophthora Sojae ◽

Knock Out ◽

Fyve Domain

Proteins that contain the FYVE zinc-finger domain are recruited to PtdIns3P-containing membranes, participating in numerous biological processes such as membrane trafficking, cytoskeletal regulation, and receptor signaling. However, the genome-wide distribution, evolution, and biological functions of FYVE-containing proteins are rarely reported for oomycetes. By genome mining of Phytophthora sojae, two proteins (PsFP1 and PsFP2) with a combination of the FYVE domain and the PX domain (a major phosphoinositide binding module) were found. To clarify the functions of PsFP1 and PsFP2, the CRISPR/Cas9-mediated gene replacement system was used to knock out the two genes respectively. Only heterozygous deletion mutants of PsFP1 were recovered, and the expression level of PsFP1 in the heterozygous knockout transformants was significantly down-regulated. These PsFP1 mutants showed a decrease in mycelial growth and pathogenicity and were more sensitive to hydrogen peroxide. These phenotypes were recovered to the level of wild-type by overexpression PsFP1 gene in the PsFP1 heterozygous knockout transformant. In contrast, deletion of PsFP2 had no significant effect on vegetative growth, asexual and sexual reproduction, pathogenicity, or oxidative stress sensitivity. PsFP1 was primarily localized in vesicle-like structures and both the FYVE and PX domains are important for its localization. Overall, our results indicate that PsFP1 plays an important role in the vegetative growth and virulence of P. sojae.

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BECLIN1: Protein Structure, Function and Regulation

Cells ◽

10.3390/cells10061522 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1522

Author(s):

Sharon Tran ◽

W. Douglas Fairlie ◽

Erinna F. Lee

Keyword(s):

Protein Structure ◽

Structure Function ◽

Membrane Trafficking ◽

Biochemical Properties ◽

Class Iii ◽

Form Class ◽

Protein Interactome ◽

Protein Functions ◽

Phosphoinositide 3 Kinase ◽

Shed Light

BECLIN1 is a well-established regulator of autophagy, a process essential for mammalian survival. It functions in conjunction with other proteins to form Class III Phosphoinositide 3-Kinase (PI3K) complexes to generate phosphorylated phosphatidylinositol (PtdIns), lipids essential for not only autophagy but other membrane trafficking processes. Over the years, studies have elucidated the structural, biophysical, and biochemical properties of BECLIN1, which have shed light on how this protein functions to allosterically regulate these critical processes of autophagy and membrane trafficking. Here, we review these findings and how BECLIN1’s diverse protein interactome regulates it, as well as its impact on organismal physiology.

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Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22158165 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8165

Author(s):

Amanda Chantziou ◽

Kostas Theodorakis ◽

Hara Polioudaki ◽

Eelco de Bree ◽

Marilena Kampa ◽

...

Keyword(s):

Breast Cancer ◽

Plasma Membrane ◽

Subcellular Localization ◽

Cancer Cells ◽

Membrane Trafficking ◽

Breast Cancer Cells ◽

Endocytic Pathway ◽

Cell Periphery ◽

Wild Type ◽

Mcf 7

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

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Sterol and lipid trafficking in mammalian cells

Biochemical Society Transactions ◽

10.1042/bst0340335 ◽

2006 ◽

Vol 34 (3) ◽

pp. 335-339 ◽

Cited By ~ 39

Author(s):

F.R. Maxfield ◽

M. Mondal

Keyword(s):

Membrane Trafficking ◽

Mammalian Cells ◽

Intracellular Transport ◽

Vesicular Transport ◽

Cholesterol Content ◽

Cellular Functions ◽

Lipid Trafficking ◽

Sterol Transport ◽

A Cell ◽

Asymmetrically Distributed

The pathways involved in the intracellular transport and distribution of lipids in general, and sterols in particular, are poorly understood. Cholesterol plays a major role in modulating membrane bilayer structure and important cellular functions, including signal transduction and membrane trafficking. Both the overall cholesterol content of a cell, as well as its distribution in specific organellar membranes are stringently regulated. Several diseases, many of which are incurable at present, have been characterized as results of impaired cholesterol transport and/or storage in the cells. Despite their importance, many fundamental aspects of intracellular sterol transport and distribution are not well understood. For instance, the relative roles of vesicular and non-vesicular transport of cholesterol have not yet been fully determined, nor are the non-vesicular transport mechanisms well characterized. Similarly, whether cholesterol is asymmetrically distributed between the two leaflets of biological membranes, and if so, how this asymmetry is maintained, is poorly understood. In this review, we present a summary of the current understanding of these aspects of intracellular trafficking and distribution of lipids, and more specifically, of sterols.

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Characterisation of an antimicrobial and phytotoxic ribonuclease secreted by the fungal wheat pathogen Zymoseptoria tritici

10.1101/130393 ◽

2017 ◽

Cited By ~ 1

Author(s):

Graeme J. Kettles ◽

Carlos Bayon ◽

Caroline A. Sparks ◽

Gail Canning ◽

Kostya Kanyuka ◽

...

Keyword(s):

Expression Pattern ◽

Effector Proteins ◽

Yeast Cells ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

List Type ◽

Expression Systems ◽

Zymoseptoria Tritici ◽

Loop Region

Abstract-The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Z. tritici secretes many functionally uncharacterised effector proteins during infection. Here we characterised a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern.-Transient expression systems were used to characterise Zt6, and mutants thereof, in both host and non-host plants. Cell-free protein expression systems monitored impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi.-We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22-amino acid N-terminal “loop” region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells but not to Z. tritici itself.-Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggests that whilst it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.

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