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1.
J Cell Biol ; 192(5): 855-71, 2011 Mar 07.
Article in English | MEDLINE | ID: mdl-21383080

ABSTRACT

Centrosomes are closely associated with the nuclear envelope (NE) throughout the cell cycle and this association is maintained in prophase when they separate to establish the future mitotic spindle. At this stage, the kinetochore constituents CENP-F, NudE, NudEL, dynein, and dynactin accumulate at the NE. We demonstrate here that the N-terminal domain of the nuclear pore complex (NPC) protein Nup133, although largely dispensable for NPC assembly, is required for efficient anchoring of the dynein/dynactin complex to the NE in prophase. Nup133 exerts this function through an interaction network via CENP-F and NudE/EL. We show that this molecular chain is critical for maintaining centrosome association with the NE at mitotic entry and contributes to this process without interfering with the previously described RanBP2-BICD2-dependent pathway of centrosome anchoring. Finally, our study reveals that tethering of centrosomes to the nuclear surface at the G2/M transition contributes, along with other cellular mechanisms, to early stages of bipolar spindle assembly.


Subject(s)
Centrosome/metabolism , Nuclear Envelope/metabolism , Nuclear Pore Complex Proteins/physiology , Nuclear Pore/metabolism , Prophase , Carrier Proteins/metabolism , Carrier Proteins/physiology , Cell Line, Tumor , Cell Polarity , Centrosome/ultrastructure , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/physiology , Dynactin Complex , Dyneins/metabolism , HeLa Cells , Humans , Intranuclear Space/metabolism , Intranuclear Space/ultrastructure , Microfilament Proteins/metabolism , Microfilament Proteins/physiology , Microtubule-Associated Proteins/metabolism , Minor Histocompatibility Antigens , Nuclear Envelope/ultrastructure , Nuclear Pore Complex Proteins/chemistry , Nuclear Pore Complex Proteins/metabolism , Protein Interaction Mapping , Spindle Apparatus/metabolism
2.
J Cell Biol ; 189(5): 795-811, 2010 May 31.
Article in English | MEDLINE | ID: mdl-20498018

ABSTRACT

The biogenesis of nuclear pore complexes (NPCs) represents a paradigm for the assembly of high-complexity macromolecular structures. So far, only three integral pore membrane proteins are known to function redundantly in NPC anchoring within the nuclear envelope. Here, we describe the identification and functional characterization of Pom33, a novel transmembrane protein dynamically associated with budding yeast NPCs. Pom33 becomes critical for yeast viability in the absence of a functional Nup84 complex or Ndc1 interaction network, which are two core NPC subcomplexes, and associates with the reticulon Rtn1. Moreover, POM33 loss of function impairs NPC distribution, a readout for a subset of genes required for pore biogenesis, including members of the Nup84 complex and RTN1. Consistently, we show that Pom33 is required for normal NPC density in the daughter nucleus and for proper NPC biogenesis and/or stability in the absence of Nup170. We hypothesize that, by modifying or stabilizing the nuclear envelope-NPC interface, Pom33 may contribute to proper distribution and/or efficient assembly of nuclear pores.


Subject(s)
Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Active Transport, Cell Nucleus/genetics , Amino Acid Sequence , Cell Proliferation , Endoplasmic Reticulum/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Nuclear Pore/genetics , Nuclear Pore/ultrastructure , Nuclear Pore Complex Proteins/genetics , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Phylogeny , Protein Binding/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Ribonuclease III/genetics , Ribonuclease III/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Telophase/physiology
3.
Microbiology (Reading) ; 152(Pt 3): 695-708, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16514150

ABSTRACT

Rgd1, a GTPase-activating protein, is the only known negative regulator of the Rho3 and Rho4 small GTPases in the yeast Saccharomyces cerevisiae. Rho3p and Rho4p are involved in regulating cell polarity by controlling polarized exocytosis. Co-inactivation of RGD1 and WSC1, which is a cell wall sensor-encoding gene, is lethal. Another plasma membrane sensor, Mid2p, is known to rescue the rgd1Deltawsc1Delta synthetic lethality. It has been proposed that Wsc1p and Mid2p act upstream of the protein kinase C (PKC) pathway to function as mechanosensors of cell wall stress. Analysis of the synthetic lethal phenomenon revealed that production of activated Rho3p and Rho4p leads to lethality in wsc1Delta cells. Inactivation of RHO3 or RHO4 was able to rescue the rgd1Deltawsc1Delta synthetic lethality, supporting the idea that the accumulation of GTP-bound Rho proteins, following loss of Rgd1p, is detrimental if the Wsc1 sensor is absent. In contrast, the genetic interaction between RGD1 and MID2 was not due to an accumulation of GTP-bound Rho proteins. It was proposed that simultaneous inactivation of RGD1 and WSC1 constitutively activates the PKC-mitogen-activated protein kinase (MAP kinase) pathway. Moreover, it was shown that the activity of this pathway was not involved in the synthetic lethal interaction, which suggests the existence of another mechanism. Consistent with this idea, it was found that perturbations in Rho3-mediated polarized exocytosis specifically impair the abundance and processing of Wsc1 and Mid2 proteins. Hence, it is proposed that Wsc1p participates in the regulation of a Rho3/4-dependent cellular mechanism, and that this is distinct from the role of Wsc1p in the PKC-MAP kinase pathway.


Subject(s)
GTP-Binding Proteins/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , rho GTP-Binding Proteins/metabolism , Gene Expression Regulation, Fungal , Heat-Shock Response , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
4.
Eukaryot Cell ; 4(8): 1375-86, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16087742

ABSTRACT

The protein kinase C (PKC) pathway is involved in the maintenance of cell shape and cell integrity in Saccharomyces cerevisiae. Here, we show that this pathway mediates tolerance to low pH and that the Bck1 and Slt2 proteins belonging to the mitogen-activated protein kinase cascade are essential for cell survival at low pH. The PKC pathway is activated during acidification of the extracellular environment, and this activation depends mainly on the Mid2p cell wall sensor. Rgd1p, which encodes a Rho GTPase-activating protein for the small G proteins Rho3p and Rho4p, also plays a role in low-pH response. The rgd1Delta strain is sensitive to low pH, and Rgd1p activates the PKC pathway in an acidic environment. Inactivation of both genes in the double mutant rgd1Delta mid2Delta strain renders yeast cells unable to survive at low pH as in bck1Delta and slt2Delta strains. Our data provide evidence for the existence of two distinct ways, one involving Mid2p and the other involving Rgd1p, with both converging to the cell integrity pathway to mediate low-pH tolerance in Saccharomyces cerevisiae. Nevertheless, even if Rgd1p acts on the PKC pathway, it seems that its mediating action on low-pH tolerance is not limited to this pathway. As the Mid2p amount plays a role in rgd1Delta sensitivity to low pH, Mid2p seems to act more like a molecular rheostat, controlling the level of PKC pathway activity and thus allowing phenotypical expression of RGD1 inactivation.


Subject(s)
Calcium-Binding Proteins/metabolism , GTPase-Activating Proteins/metabolism , Membrane Proteins/metabolism , Protein Kinase C/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Calcium-Binding Proteins/genetics , Cell Survival , Cell Wall , GTPase-Activating Proteins/genetics , Genotype , Hydrogen-Ion Concentration , Intracellular Signaling Peptides and Proteins , MADS Domain Proteins , Membrane Glycoproteins , Membrane Proteins/genetics , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Models, Biological , Phosphorylation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Suppression, Genetic/genetics , Temperature , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism
5.
Gene ; 351: 159-69, 2005 May 23.
Article in English | MEDLINE | ID: mdl-15922872

ABSTRACT

The RhoGAP Rgd1p is involved in different signal transduction pathways in Saccharomyces cerevisiae through its regulatory activity upon the Rho3 and Rho4 GTPases. The rgd1Delta mutant, which presents a mortality at the entry into the stationary phase in minimal medium, is sensitive to medium acidification caused by biomass augmentation. We showed that low-pH shock leads to abnormal intracellular acidification of the rgd1Delta mutant. Transcriptional regulation of RGD1 was studied in several stress conditions and we observed an activation of RGD1 transcription at low pH and after heat and oxidative shocks. The transcription level at low pH and after heat shock was demonstrated to depend on the STRE box located in the RGD1 promoter. The general stress-activated transcription factors Msn2p and Msn4p as well as the HOG pathway were shown to mainly act on the basal RGD1 transcriptional level in normal and stress conditions.


Subject(s)
DNA-Binding Proteins/metabolism , GTPase-Activating Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transcription Factors/metabolism , Binding Sites/genetics , Culture Media/pharmacology , DNA-Binding Proteins/genetics , GTPase-Activating Proteins/genetics , Gene Expression Regulation, Fungal/drug effects , Hydrochloric Acid/pharmacology , Hydrogen-Ion Concentration , Lac Operon/genetics , Mutation , Phenotype , Plasmids/genetics , Promoter Regions, Genetic/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Time Factors , Transcription Factors/genetics , Transcription, Genetic/drug effects , beta-Galactosidase/metabolism
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