Antagonistic regulation of Fus2p nuclear localization by pheromone signaling and the cell cycle

When yeast cells sense mating pheromone, they undergo a characteristic response involving changes in transcription, cell cycle arrest in early G1, and polarization along the pheromone gradient. Cells in G2/M respond to pheromone at the transcriptional level but do not polarize or mate until G1. Fus2p, a key regulator of cell fusion, localizes to the tip of the mating projection during pheromone-induced G1 arrest. Although Fus2p was expressed in G2/M cells after pheromone induction, it accumulated in the nucleus until after cell division. As cells arrested in G1, Fus2p was exported from the nucleus and localized to the nascent tip. Phosphorylation of Fus2p by Fus3p was required for Fus2p export; cyclin/Cdc28p-dependent inhibition of Fus3p during late G1 through S phase was sufficient to block exit. However, during G2/M, when Fus3p was activated by pheromone signaling, Cdc28p activity again blocked Fus2p export. Our results indicate a novel mechanism by which pheromone-induced proteins are regulated during the transition from mitosis to conjugation

Nerve Growth Factor Stimulates Interaction of Cayman Ataxia Protein BNIP-H/Caytaxin with Peptidyl-Prolyl Isomerase Pin1 in Differentiating Neurons

Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin) lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation. (213 words

A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

BACKGROUND: RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. CONCLUSIONS/SIGNIFICANCE: A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking

On a Model for the Prediction of the Friction Coefficient in Mixed Lubrication Based on a Load-Sharing Concept with Measured Surface Roughness

A new model was developed for the simulation of the friction coefficient in lubricated sliding line contacts. A half-space-based contact algorithm was linked with a numerical elasto-hydrodynamic lubrication solver using the load-sharing concept. The model was compared with an existing asperity-based friction model for a set of theoretical simulations. Depending on the load and surface roughness, the difference in friction varied up to 32 %. The numerical lubrication model makes it possible to also calculate lightly loaded contacts and can easily be extended to solve transient problems. Experimental validation was performed by measuring the friction coefficient as a function of sliding velocity for the stationary case

The transition to adhesive wear of lubricated concentrated contacts

Validation measurements are carried out by means of pin-on-disk measurements, in which the pin consists of a cylindrical element, resulting in a line contact situation in contact with the disk. For these measurements a special high load pin-on-disk tribotester has been developed. From the experiments can be concluded that the measured transitions are well predicted by the model which is developed in this thesis, in contrast to the calculation models known from literature

Determination of variator robustness under macro slip conditions for a push belt CVT

Developments in clamping force control for the push belt Continuously Variable Transmission (CVT) aim at increased efficiency in combination with improved robustness. Current control strategies attempt to prevent macro slip between elements and pulleys at all times for maximum robustness. In order to search for the limiting factors in developing a new control strategy, situations where macro slip occurs have been investigated. New tribological insights show that macro slip is acceptable to a certain extent. An important failure mechanism proves to be the occurrence of adhesive wear in the contact leading to a loss in torque transfer. As a combination of normal load and slip speed, respectively clamping force and slip rate in a variator, a transition can be found from a safe wear region to an excessive/adhesive wear region. The occurrence of this transition has been verified with experiments on CVT variator level. New applications based on current understanding are found in the field of new control strategies