Superdiffusion in a Model for Diffusion in a Molecularly Crowded Environment

We present a model for diffusion in a molecularly crowded environment. The model consists of random barriers in percolation network. Random walks in the presence of slowly moving barriers show normal diffusion for long times, but anomalous diffusion at intermediate times. The effective exponents for square distance versus time usually are below one at these intermediate times, but can be also larger than one for high barrier concentrations. Thus we observe sub- as well as super-diffusion in a crowded environment.Comment: 8 pages including 4 figure

Specificity of Transmembrane Protein Palmitoylation in Yeast

Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called “Asp-His-His-Cys- Cysteine Rich Domain” (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether

The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms

© 2008 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0).The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3β homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.Deutsche Forschungsgemeinschaf

Modeling Robustness Tradeoffs in Yeast Cell Polarization Induced by Spatial Gradients

Cells localize (polarize) internal components to specific locations in response to external signals such as spatial gradients. For example, yeast cells form a mating projection toward the source of mating pheromone. There are specific challenges associated with cell polarization including amplification of shallow external gradients of ligand to produce steep internal gradients of protein components (e.g. localized distribution), response over a broad range of ligand concentrations, and tracking of moving signal sources. In this work, we investigated the tradeoffs among these performance objectives using a generic model that captures the basic spatial dynamics of polarization in yeast cells, which are small. We varied the positive feedback, cooperativity, and diffusion coefficients in the model to explore the nature of this tradeoff. Increasing the positive feedback gain resulted in better amplification, but also produced multiple steady-states and hysteresis that prevented the tracking of directional changes of the gradient. Feedforward/feedback coincidence detection in the positive feedback loop and multi-stage amplification both improved tracking with only a modest loss of amplification. Surprisingly, we found that introducing lateral surface diffusion increased the robustness of polarization and collapsed the multiple steady-states to a single steady-state at the cost of a reduction in polarization. Finally, in a more mechanistic model of yeast cell polarization, a surface diffusion coefficient between 0.01 and 0.001 µm2/s produced the best polarization performance, and this range is close to the measured value. The model also showed good gradient-sensitivity and dynamic range. This research is significant because it provides an in-depth analysis of the performance tradeoffs that confront biological systems that sense and respond to chemical spatial gradients, proposes strategies for balancing this tradeoff, highlights the critical role of lateral diffusion of proteins in the membrane on the robustness of polarization, and furnishes a framework for future spatial models of yeast cell polarization

An Abp1-Dependent Route of Endocytosis Functions when the Classical Endocytic Pathway in Yeast Is Inhibited

Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the ‘classic’ pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the ‘classic’ pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions

Spatial Modeling of Vesicle Transport and the Cytoskeleton: The Challenge of Hitting the Right Road

The membrane trafficking machinery provides a transport and sorting system for many cellular proteins. We propose a mechanistic agent-based computer simulation to integrate and test the hypothesis of vesicle transport embedded into a detailed model cell. The method tracks both the number and location of the vesicles. Thus both the stochastic properties due to the low numbers and the spatial aspects are preserved. The underlying molecular interactions that control the vesicle actions are included in a multi-scale manner based on the model of Heinrich and Rapoport (2005). By adding motor proteins we can improve the recycling process of SNAREs and model cell polarization. Our model also predicts that coat molecules should have a high turnover at the compartment membranes, while the turnover of motor proteins has to be slow. The modular structure of the underlying model keeps it tractable despite the overall complexity of the vesicle system. We apply our model to receptor-mediated endocytosis and show how a polarized cytoskeleton structure leads to polarized distributions in the plasma membrane both of SNAREs and the Ste2p receptor in yeast. In addition, we can couple signal transduction and membrane trafficking steps in one simulation, which enables analyzing the effect of receptor-mediated endocytosis on signaling

The Ankyrin Repeats and DHHC S-acyl Transferase Domain of AKR1 Act Independently to Regulate Switching from Vegetative to Mating States in Yeast

Signal transduction from G-protein coupled receptors to MAPK cascades through heterotrimeric G-proteins has been described for many eukaryotic systems. One of the best-characterised examples is the yeast pheromone response pathway, which is negatively regulated by AKR1. AKR1-like proteins are present in all eukaryotes and contain a DHHC domain and six ankyrin repeats. Whilst the DHHC domain dependant S-acyl transferase (palmitoyl transferase) function of AKR1 is well documented it is not known whether the ankyrin repeats are also required for this activity. Here we show that the ankyrin repeats of AKR1 are required for full suppression of the yeast pheromone response pathway, by sequestration of the Gβγ dimer, and act independently of AKR1 S-acylation function. Importantly, the functions provided by the AKR1 ankyrin repeats and DHHC domain are not required on the same molecule to fully restore WT phenotypes and function. We also show that AKR1 molecules are S-acylated at locations other than the DHHC cysteine, increasing the abundance of AKR1 in the cell. Our results have important consequences for studies of AKR1 function, including recent attempts to characterise S-acylation enzymology and kinetics. Proteins similar to AKR1 are found in all eukaryotes and our results have broad implications for future work on these proteins and the control of switching between Gβγ regulated pathways

Putative DHHC-Cysteine-Rich Domain S-Acyltransferase in Plants

Protein S-acyltransferases (PATs) containing Asp-His-His-Cys within a Cys-rich domain (DHHC-CRD) are polytopic transmembrane proteins that are found in eukaryotic cells and mediate the S-acylation of target proteins. S-acylation is an important secondary and reversible modification that regulates the membrane association, trafficking and function of target proteins. However, little is known about the characteristics of PATs in plants. Here, we identified 804 PATs from 31 species with complete genomes. The analysis of the phylogenetic relationships suggested that all of the PATs fell into 8 groups. In addition, we analysed the phylogeny, genomic organization, chromosome localisation and expression pattern of PATs in Arabidopsis, Oryza sative, Zea mays and Glycine max. The microarray data revealed that PATs genes were expressed in different tissues and during different life stages. The preferential expression of the ZmPATs in specific tissues and the response of Zea mays to treatments with phytohormones and abiotic stress demonstrated that the PATs play roles in plant growth and development as well as in stress responses. Our data provide a useful reference for the identification and functional analysis of the members of this protein family

Singularity in polarization:rewiring yeast cells to make two buds

SummaryFor budding yeast to ensure formation of only one bud, cells must polarize toward one, and only one, site. Polarity establishment involves the Rho family GTPase Cdc42, which concentrates at polarization sites via a positive feedback loop. To assess whether singularity is linked to the specific Cdc42 feedback loop, we disabled the yeast cell's endogenous amplification mechanism and synthetically rewired the cells to employ a different positive feedback loop. Rewired cells violated singularity, occasionally making two buds. Even cells that made only one bud sometimes initiated two clusters of Cdc42, but then one cluster became dominant. Mathematical modeling indicated that, given sufficient time, competition between clusters would promote singularity. In rewired cells, competition occurred slowly and sometimes failed to develop a single “winning” cluster before budding. Slowing competition in normal cells also allowed occasional formation of two buds, suggesting that singularity is enforced by rapid competition between Cdc42 clusters

Reversible, cooperative reactions of yeast vacuole docking

Homotypic yeast vacuole fusion occurs in three stages: (i) priming reactions, which are independent of vacuole clustering, (ii) docking, in which vacuoles cluster and accumulate fusion proteins and fusion regulatory lipids at a ring-shaped microdomain surrounding the apposed membranes of each docked vacuole, where fusion will occur, and (iii) bilayer fusion/compartment mixing. These stages require vacuolar SNAREs, SNARE-chaperones, GTPases, effector complexes, and chemically minor but functionally important lipids. For each, we have developed specific ligands that block fusion and conditions that reverse each block. Using them, we test whether docking entails a linearly ordered series of catalytic events, marked by sequential acquisition of resistance to inhibitors, or whether docking subreactions are cooperative and/or reversible. We find that each fusion protein and regulatory lipid is needed throughout docking, indicative of a reversible or highly cooperative assembly of the fusion-competent vertex ring. In accord with this cooperativity, vertices enriched in one fusion catalyst are enriched in others. Docked vacuoles finally assemble SNARE complexes, yet still require physiological temperature and lipid rearrangements to complete fusion