Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

AbstractThe tubulin-like GTPase protein FtsZ, which forms a discontinuous cytokinetic ring at mid-cell, is a central player to recruit the division machinery to orchestrate cell division. To guarantee the production of two identical daughter cells, the assembly of FtsZ, namely Z-ring, and its precise positioning should be finely regulated. In Streptococcus pneumoniae, the positioning of Z-ring at the division site is mediated by a bitopic membrane protein MapZ (mid-cell-anchored protein Z) through direct interactions between the intracellular domain (termed MapZ-N (the intracellular domain of MapZ)) and FtsZ. Using nuclear magnetic resonance titration experiments, we clearly assigned the key residues involved in the interactions. In the presence of MapZ-N, FtsZ gains a shortened activation delay, a lower critical concentration for polymerization and a higher cooperativity towards GTP hydrolysis. On the other hand, MapZ-N antagonizes the lateral interactions of single-stranded filaments of FtsZ, thus slows down the formation of highly bundled FtsZ polymers and eventually maintains FtsZ at a dynamic state. Altogether, we conclude that MapZ is not only an accelerator to trigger the polymerization of FtsZ, but also a brake to tune the velocity to form the end-product, FtsZ bundles. These findings suggest that MapZ is a multi-functional regulator towards FtsZ that controls both the precise positioning and proper timing of FtsZ polymerization.

Download Full-text

Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase

The Journal of Cell Biology ◽

10.1083/jcb.200408087 ◽

2005 ◽

Vol 168 (2) ◽

pp. 209-219 ◽

Cited By ~ 57

Author(s):

Félix Machín ◽

Jordi Torres-Rosell ◽

Adam Jarmuz ◽

Luis Aragón

Keyword(s):

Cell Division ◽

Ribosomal Dna ◽

Budding Yeast ◽

Mitotic Cell ◽

Yeast Genome ◽

Daughter Cells ◽

Late Anaphase ◽

Mother And Daughter ◽

The Right ◽

Chromosome Resolution

Mitotic cell division involves the equal segregation of all chromosomes during anaphase. The presence of ribosomal DNA (rDNA) repeats on the right arm of chromosome XII makes it the longest in the budding yeast genome. Previously, we identified a stage during yeast anaphase when rDNA is stretched across the mother and daughter cells. Here, we show that resolution of sister rDNAs is achieved by unzipping of the locus from its centromere-proximal to centromere-distal regions. We then demonstrate that during this stretched stage sister rDNA arrays are neither compacted nor segregated despite being largely resolved from each other. Surprisingly, we find that rDNA segregation after this period no longer requires spindles but instead involves Cdc14-dependent rDNA axial compaction. These results demonstrate that chromosome resolution is not simply a consequence of compacting chromosome arms and that overall rDNA compaction is necessary to mediate the segregation of the long arm of chromosome XII.

Download Full-text

Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801287115 ◽

2018 ◽

Vol 115 (23) ◽

pp. E5279-E5288 ◽

Cited By ~ 24

Author(s):

Minji Lee ◽

Jong Hyun Kim ◽

Ina Yoon ◽

Chulho Lee ◽

Mohammad Fallahi Sichani ◽

...

Keyword(s):

Protein Synthesis ◽

Trna Synthetase ◽

Gtp Hydrolysis ◽

Mtorc1 Signaling ◽

Amino Acid Sensing ◽

Gtpase Cycle ◽

Rag Gtpase ◽

Cancer Tissues ◽

Mtorc1 Activation ◽

Hydrolysis Of

A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating “ON” switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an “OFF” switch by controlling GTP hydrolysis of RagB in the Rag GTPase–mTORC1 axis. The LRS–RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP–GDP cycle of the RagD–RagB pair, rather than the RagC–RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD–RagB pair can overcome the absence of the RagC–RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD–RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.

Download Full-text

Selenocysteine Inserting RNA Elements Modulate GTP Hydrolysis of Elongation Factor SelB†

Biochemistry ◽

10.1021/bi972298k ◽

1998 ◽

Vol 37 (3) ◽

pp. 885-890 ◽

Cited By ~ 38

Author(s):

Alexander Hüttenhofer ◽

August Böck

Keyword(s):

Elongation Factor ◽

Gtp Hydrolysis ◽

Rna Elements ◽

Hydrolysis Of

Download Full-text

SmpB Triggers GTP Hydrolysis of Elongation Factor Tu on Ribosomes by Compensating for the Lack of Codon-Anticodon Interaction during Trans-translation Initiation

Journal of Biological Chemistry ◽

10.1074/jbc.m512165200 ◽

2006 ◽

Vol 281 (23) ◽

pp. 15987-15996 ◽

Cited By ~ 23

Author(s):

Yoshihiro Shimizu ◽

Takuya Ueda

Keyword(s):

Translation Initiation ◽

Elongation Factor ◽

Elongation Factor Tu ◽

Gtp Hydrolysis ◽

Hydrolysis Of

Download Full-text

GTP Hydrolysis of TC10 Promotes Neurite Outgrowth through Exocytic Fusion of Rab11- and L1-Containing Vesicles by Releasing Exocyst Component Exo70

PLoS ONE ◽

10.1371/journal.pone.0079689 ◽

2013 ◽

Vol 8 (11) ◽

pp. e79689 ◽

Cited By ~ 30

Author(s):

Akane Fujita ◽

Shingo Koinuma ◽

Sayaka Yasuda ◽

Hiroyuki Nagai ◽

Hiroyuki Kamiguchi ◽

...

Keyword(s):

Neurite Outgrowth ◽

Gtp Hydrolysis ◽

Hydrolysis Of

Download Full-text

Lysine and Polyamines Are Substrates for Transglutamination of Rho by the BordetellaDermonecrotic Toxin

Infection and Immunity ◽

10.1128/iai.69.12.7663-7670.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7663-7670 ◽

Cited By ~ 16

Author(s):

Gudula Schmidt ◽

Udo-Michael Goehring ◽

Joerg Schirmer ◽

Sandrine Uttenweiler-Joseph ◽

Matthias Wilm ◽

...

Keyword(s):

Rho Gtpases ◽

Affinity Purification ◽

Dependent Manner ◽

Rho Proteins ◽

Concentration Dependent Manner ◽

Gtp Hydrolysis ◽

Dermonecrotic Toxin ◽

Hydrolysis Of ◽

Active Fragment

ABSTRACT Bordetella dermonecrotic toxin (DNT) catalyzes the transglutamination of glutamine-63/61 of Rho GTPases, thereby constitutively activating Rho proteins. Here we identified second substrates for transglutamination of RhoA by DNT. The enzymatically active fragment of DNT (residues 1136 to 1451, ΔDNT) induced the incorporation of l-[14C]lysine in RhoA in a concentration-dependent manner. Also, Rac and Cdc42, but not Ras, were transglutaminated with lysine by ΔDNT. Transglutamination of the GTPase with l-lysine inhibited intrinsic and Rho-GAP-stimulated GTP hydrolysis of RhoA. In contrast to lysine, treatment of RhoA with alanine, arginine, and glutamine were not able to substitute for lysine in the transglutamination reaction. DNT increased the incorporation of l-[14C]lysine into embryonic bovine lung cells. Microinjection of GST-RhoA together with the enzymatically active DNT fragment intoXenopus oocytes, subsequent affinity purification of modified GST-RhoA, and mass spectrometry identified attachment of putrescine or spermidine at glutamine-63 of RhoA. A comparison of putrescine, spermidine, and lysine as substrates for DNT-induced transglutamination of RhoA revealed that lysine is a preferred second substrate at least in vitro.

Download Full-text

Bacterial dynamin-like protein DynA mediates lipid and content mixing and shows phospholipid specificity

10.1101/363689 ◽

2018 ◽

Author(s):

Lijun Guo ◽

Marc Bramkamp

Keyword(s):

Resonance Energy Transfer ◽

Resonance Energy ◽

Membrane Stress ◽

Gtp Hydrolysis ◽

Cellular Processes ◽

Organelle Division ◽

Membrane Tethering ◽

Hydrolysis Of

ABSTRACTThe dynamins family of GTPases is involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. The GTP hydrolysis cycle of dynamin translates to a conformational change in the protein structure, which forces the underlying lipid layer into an energetically unstable conformation that promotes membrane rearrangements. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. In recent years, our group has reported that the dynamin-like protein DynA from Bacillus subtilis mediates nucleotide-independent membrane tethering in vitro and contributes to the innate immunity of bacteria against membrane stress and phage infection. However, so far the mechanism of membrane stress response and the role of GTP hydrolysis remain unclear. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if the dynamin-like protein DynA from B. subtilis induces membrane full fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer (FRET) indicated that DynA could induce aqueous content mixing even in absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow response. Surprisingly, digestion of protein mediated an instantl rise of content exchange, supporting the assumption that disassembly of DynA is the fundamental power for fusion-through-hemifusion.

Download Full-text

Analysis of Cell Cycle Progression in the Budding Yeast S. cerevisiae

Methods in Molecular Biology - Cell Cycle Oscillators ◽

10.1007/978-1-0716-1538-6_19 ◽

2021 ◽

pp. 265-276

Author(s):

Deniz Pirincci Ercan ◽

Frank Uhlmann

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Cell Cycle Progression ◽

Budding Yeast ◽

Expression Profiles ◽

Model Organisms ◽

Cyclin Dependent Kinase ◽

Cycle Progression ◽

Daughter Cells ◽

Substrate Phosphorylation

AbstractThe cell cycle is an ordered series of events by which cells grow and divide to give rise to two daughter cells. In eukaryotes, cyclin–cyclin-dependent kinase (cyclin–Cdk) complexes act as master regulators of the cell division cycle by phosphorylating numerous substrates. Their activity and expression profiles are regulated in time. The budding yeast S. cerevisiae was one of the pioneering model organisms to study the cell cycle. Its genetic amenability continues to make it a favorite model to decipher the principles of how changes in cyclin-Cdk activity translate into the intricate sequence of substrate phosphorylation events that govern the cell cycle. In this chapter, we introduce robust and straightforward methods to analyze cell cycle progression in S. cerevisiae. These techniques can be utilized to describe cell cycle events and to address the effects of perturbations on accurate and timely cell cycle progression.

Download Full-text