Sac1 Lipid Phosphatase and Stt4 Phosphatidylinositol 4-Kinase Regulate a Pool of Phosphatidylinositol 4-Phosphate That Functions in the Control of the Actin Cytoskeleton and Vacuole Morphology

Synthesis and turnover of phosphoinositides are tightly regulated processes mediated by a set of recently identified kinases and phosphatases. We analyzed the primary role of the phosphoinositide phosphatase Sac1p in Saccharomyces cerevisiae with the use of a temperature-sensitive allele of this gene. Our analysis demonstrates that inactivation of Sac1p leads to a specific increase in the cellular levels of phosphatidylinositol 4-phosphate (PtdIns(4)P), accompanied by changes in vacuole morphology and an accumulation of lipid droplets. We have found that the majority of Sac1p localizes to the endoplasmic reticulum, and this localization is crucial for the efficient turnover of PtdIns(4)P. By generating double mutant strains harboring the sac1tsallele and one of two temperature-sensitive PtdIns 4-kinase genes,stt4tsor pik1ts, we have demonstrated that the bulk of PtdIns(4)P that accumulates insac1 mutant cells is generated by the Stt4 PtdIns 4-kinase, and not Pik1p. Consistent with these findings, inactivation of Sac1p partially rescued defects associated withstt4tsbut notpik1tsmutant cells. To analyze potential overlapping functions between Sac1p and other homologous phosphoinositide phosphatases, sac1tsmutant cells lacking various other synaptojanin-like phosphatases were generated. These double and triple mutants exacerbated the accumulation of intracellular phosphoinositides and caused defects in Golgi function. Together, our results demonstrate that Sac1p primarily turns over Stt4p-generated PtdIns(4)P and that the membrane localization of Sac1p is important for its function in vivo. Regulation of this PtdIns(4)P pool appears to be crucial for the maintenance of vacuole morphology, regulation of lipid storage, Golgi function, and actin cytoskeleton organization.

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The Saccharomyces cerevisiae SDA1 gene is required for actin cytoskeleton organization and cell cycle progression

Journal of Cell Science ◽

10.1242/jcs.113.7.1199 ◽

2000 ◽

Vol 113 (7) ◽

pp. 1199-1211

Author(s):

G. Buscemi ◽

F. Saracino ◽

D. Masnada ◽

M.L. Carbone

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Actin Cytoskeleton ◽

Cell Cycle Progression ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Cycle Progression ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiae novel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G(1) by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1 temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1 or in cdc28-13 cells released from G(1) block, prevents recovery of budding and DNA replication. We also show that the block in G(1) caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G(1) and that Sda1 is involved in the control of the actin cytoskeleton.

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Regulation of Yeast Actin Cytoskeleton-Regulatory Complex Pan1p/Sla1p/End3p by Serine/Threonine Kinase Prk1p

Molecular Biology of the Cell ◽

10.1091/mbc.12.12.3759 ◽

2001 ◽

Vol 12 (12) ◽

pp. 3759-3772 ◽

Cited By ~ 72

Author(s):

Guisheng Zeng ◽

Xianwen Yu ◽

Mingjie Cai

Keyword(s):

Actin Cytoskeleton ◽

Regulatory Protein ◽

Strong Support ◽

Stable Complex ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

The serine/threonine kinase Prk1p is known to be involved in the regulation of the actin cytoskeleton organization in budding yeast. One possible function of Prk1p is the negative regulation of Pan1p, an actin patch regulatory protein that forms a complex in vivo with at least two other proteins, Sla1p and End3p. In this report, we identified Sla1p as another substrate for Prk1p. The phosphorylation of Sla1p by Prk1p was established in vitro with the use of immunoprecipitated Prk1p and in vivo with the use ofPRK1 overexpression, and was further supported by the finding that immunoprecipitated Sla1p contained PRK1- and ARK1-dependent kinase activities. Stable complex formation between Prk1p and Sla1p/Pan1p in vivo could be observed once the phosphorylation reaction was blocked by mutation in the catalytic site of Prk1p. Elevation of Prk1p activities in wild-type cells resulted in a number of deficiencies, including those in colocalization of Pan1p and Sla1p, endocytosis, and cell wall morphogenesis, likely attributable to a disintegration of the Pan1p/Sla1p/End3p complex. These results lend a strong support to the model that the phosphorylation of the Pan1p/Sla1p/End3p complex by Prk1p is one of the important mechanisms by which the organization and functions of the actin cytoskeleton are regulated.

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The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast.

Molecular Biology of the Cell ◽

10.1091/mbc.5.9.1023 ◽

1994 ◽

Vol 5 (9) ◽

pp. 1023-1037 ◽

Cited By ~ 182

Author(s):

H Bénédetti ◽

S Raths ◽

F Crausaz ◽

H Riezman

Keyword(s):

Actin Cytoskeleton ◽

Protein Function ◽

Consensus Sequence ◽

Growth Defect ◽

Temperature Sensitive ◽

Cytoskeleton Organization ◽

Terminal Domain ◽

C Terminus ◽

Ef Hand ◽

Actin Cytoskeleton Organization

Two Saccharomyces cerevisiae mutants, end3 and end4, defective in the internalization step of endocytosis, have previously been isolated. The END3 gene was cloned by complementation of the temperature-sensitive growth defect caused by the end3 mutation and the END3 nucleotide sequence was determined. The END3 gene product is a 40-kDa protein that has a putative EF-hand Ca(2+)-binding site, a consensus sequence for the binding of phosphotidylinositol 4,5-bisphosphate (PIP2), and a C-terminal domain containing two homologous regions of 17-19 aa. The EF-hand consensus and the putative PIP2-binding sites are seemingly not required for End3 protein function. In contrast, different portions of the End3p N-terminal domain, and at least one of the two repeated regions in its C-terminus, are required for End3p activity. Disruption of the END3 gene yielded cells with the same phenotype as the original end3 mutant. An end3ts allele was obtained and this allowed us to demonstrate that End3p is specifically involved in the internalization step of endocytosis. In addition, End3p was shown to be required for proper organization of the actin cytoskeleton and for the correct distribution of chitin at the cell surface.

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Gα12/13 regulate epiboly by inhibiting E-cadherin activity and modulating the actin cytoskeleton

The Journal of Cell Biology ◽

10.1083/jcb.200805148 ◽

2009 ◽

Vol 184 (6) ◽

pp. 909-921 ◽

Cited By ~ 48

Author(s):

Fang Lin ◽

Songhai Chen ◽

Diane S. Sepich ◽

Jennifer Ray Panizzi ◽

Sherry G. Clendenon ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Molecular Mechanisms ◽

Genetic Studies ◽

Yolk Cell ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization ◽

Cell Layers ◽

Dependent Pathway ◽

E Cadherin

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation, and involves coordinated movements of several cell layers. Although recent studies have begun to elucidate the processes that underlie these epibolic movements, the cellular and molecular mechanisms involved remain to be fully defined. Here, we show that gastrulae with altered Gα12/13 signaling display delayed epibolic movement of the deep cells, abnormal movement of dorsal forerunner cells, and dissociation of cells from the blastoderm, phenocopying e-cadherin mutants. Biochemical and genetic studies indicate that Gα12/13 regulate epiboly, in part by associating with the cytoplasmic terminus of E-cadherin, and thereby inhibiting E-cadherin activity and cell adhesion. Furthermore, we demonstrate that Gα12/13 modulate epibolic movements of the enveloping layer by regulating actin cytoskeleton organization through a RhoGEF/Rho-dependent pathway. These results provide the first in vivo evidence that Gα12/13 regulate epiboly through two distinct mechanisms: limiting E-cadherin activity and modulating the organization of the actin cytoskeleton.

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A SAC phosphoinositide phosphatase controls rice development via hydrolyzing phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate

10.1101/740001 ◽

2019 ◽

Author(s):

Tao Guo ◽

Hua-Chang Chen ◽

Zi-Qi Lu ◽

Min Diao ◽

Ke Chen ◽

...

Keyword(s):

Actin Polymerization ◽

Membrane Lipids ◽

Dependent Manner ◽

Cytoskeleton Organization ◽

Cellular Processes ◽

Phosphoinositide Phosphatase ◽

Actin Cytoskeleton Organization ◽

Phosphatidylinositol 4 ◽

Related Proteins

AbstractPhosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PIs-dependent modulation remain largely elusive, the effects of disturbed PIs metabolism could be employed to propose regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT 1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice. Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related proteins 2 and 3 (Arp2/3) complex-nucleated actin branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effect in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulted from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering the cell development. These findings imply that Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing new insights into the relationship between PIs homeostasis and plants growth and development.

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A Mutation in dVps28 Reveals a Link between a Subunit of the Endosomal Sorting Complex Required for Transport-I Complex and the Actin Cytoskeleton in Drosophila

Molecular Biology of the Cell ◽

10.1091/mbc.e04-11-1013 ◽

2005 ◽

Vol 16 (5) ◽

pp. 2301-2312 ◽

Cited By ~ 29

Author(s):

Evgueni A. Sevrioukov ◽

Nabil Moghrabi ◽

Mary Kuhn ◽

Helmut Krämer

Keyword(s):

Actin Cytoskeleton ◽

Endosomal Sorting ◽

Cytoskeleton Organization ◽

Early Embryonic Lethality ◽

Drosophila Homolog ◽

Endosomal Membranes ◽

Actin Cytoskeleton Organization ◽

A Subunit ◽

Mutant Cells ◽

Drosophila Embryos

Proteins that constitute the endosomal sorting complex required for transport (ESCRT) are necessary for the sorting of proteins into multivesicular bodies (MVBs) and the budding of several enveloped viruses, including HIV-1. The first of these complexes, ESCRT-I, consists of three proteins: Vps28p, Vps37p, and Vps23p or Tsg101 in mammals. Here, we characterize a mutation in the Drosophila homolog of vps28. The dVps28 gene is essential: homozygous mutants die at the transition from the first to second instar. Removal of maternally contributed dVps28 causes early embryonic lethality. In such embryos lacking dVps28, several processes that require the actin cytoskeleton are perturbed, including axial migration of nuclei, formation of transient furrows during cortical divisions in syncytial embryos, and the subsequent cellularization. Defects in actin cytoskeleton organization also become apparent during sperm individualization in dVps28 mutant testis. Because dVps28 mutant cells contained MVBs, these defects are unlikely to be a secondary consequence of disrupted MVB formation and suggest an interaction between the actin cytoskeleton and endosomal membranes in Drosophila embryos earlier than previously appreciated.

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Inactivation of Two Dictyostelium discoideum Genes, DdPIK1 and DdPIK2, Encoding Proteins Related to Mammalian Phosphatidylinositide 3-kinases, Results in Defects in Endocytosis, Lysosome to Postlysosome Transport, and Actin Cytoskeleton Organization

The Journal of Cell Biology ◽

10.1083/jcb.136.6.1271 ◽

1997 ◽

Vol 136 (6) ◽

pp. 1271-1286 ◽

Cited By ~ 61

Author(s):

Greg Buczynski ◽

Bryon Grove ◽

Anson Nomura ◽

Maurice Kleve ◽

John Bush ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Dictyostelium Discoideum ◽

Mutant Strain ◽

Fluorescent Microscopy ◽

Membrane Traffic ◽

Fluid Phase ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization ◽

Endosomal Pathway ◽

Mutant Cells

Phosphatidylinositide 3-kinases (PI 3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum, DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (Δddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, Δddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme α-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that Δddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1–3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, Δddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

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The Phosphatidylinositol 4,5-Biphosphate and TORC2 Binding Proteins Slm1 and Slm2 Function in Sphingolipid Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.02403-05 ◽

2006 ◽

Vol 26 (15) ◽

pp. 5861-5875 ◽

Cited By ~ 101

Author(s):

Mitsuaki Tabuchi ◽

Anjon Audhya ◽

Ainslie B. Parsons ◽

Charles Boone ◽

Scott D. Emr

Keyword(s):

Actin Cytoskeleton ◽

Essential Role ◽

Synthetic Lethality ◽

Sphingolipid Metabolism ◽

Actin Organization ◽

Cytoskeleton Organization ◽

Calcium Calmodulin Dependent ◽

Actin Cytoskeleton Organization ◽

Phosphatidylinositol 4

ABSTRACT The Stt4 phosphatidylinositol 4-kinase has been shown to generate a pool of phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, critical for actin cytoskeleton organization and cell viability. To further understand the essential role of Stt4-mediated PI4P production, we performed a genetic screen using the stt4 ts mutation to identify candidate regulators and effectors of PI4P. From this analysis, we identified several genes that have been previously implicated in lipid metabolism. In particular, we observed synthetic lethality when both sphingolipid and PI4P synthesis were modestly diminished. Consistent with these data, we show that the previously characterized phosphoinositide effectors, Slm1 and Slm2, which regulate actin organization, are also necessary for normal sphingolipid metabolism, at least in part through regulation of the calcium/calmodulin-dependent phosphatase calcineurin, which binds directly to both proteins. Additionally, we identify Isc1, an inositol phosphosphingolipid phospholipase C, as an additional target of Slm1 and Slm2 negative regulation. Together, our data suggest that Slm1 and Slm2 define a molecular link between phosphoinositide and sphingolipid signaling and thereby regulate actin cytoskeleton organization.

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