Expression and functional profiling reveal distinct gene classes involved in fatty acid metabolism

Cells respond to fatty acid exposure by metabolic reorganization and proliferation of peroxisomes. Described here is the development and application of a genome-wide screen to identify nonessential yeast genes necessary for efficient metabolism of myristic and oleic acids. Comparison of the resultant fitness data set with an integrated data set of genes transcriptionally responsive to fatty acids revealed very little overlap between the data sets. Furthermore, the fitness data set enriched for genes involved in peroxisome biogenesis and other processes related to cell morphology, whereas the expression data set enriched for genes related to metabolism. These data suggest that in response to fatty acid exposure, transcriptional control is biased towards metabolic reorganization, and structural changes tend to be controlled post-transcriptionally. They also suggest that fatty acid responsive metabolic networks are more robust than those related to cell structure. Statistical analyses of these and other global data sets suggest that the utilization of distinct control mechanisms for the execution of morphological versus metabolic responses is widespread

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

A combination of yeast genetics, synthetic genetic array analysis, and high-throughput screening reveals that sumoylation of Mcm21p promotes disassembly of the mitotic spindle

Odin (ANKS1A) Modulates EGF Receptor Recycling and Stability

<div><p>The ANKS1A gene product, also known as Odin, was first identified as a tyrosine-phosphorylated component of the epidermal growth factor receptor network. Here we show that Odin functions as an effector of EGFR recycling. In EGF-stimulated HEK293 cells tyrosine phosphorylation of Odin was induced prior to EGFR internalization and independent of EGFR-to-ERK signaling. Over-expression of Odin increased EGF-induced EGFR trafficking to recycling endosomes and recycling back to the cell surface, and decreased trafficking to lysosomes and degradation. Conversely, Odin knockdown in both HEK293 and the non-small cell lung carcinoma line RVH6849, which expresses roughly 10-fold more EGF receptors than HEK293, caused decreased EGFR recycling and accelerated trafficking to the lysosome and degradation. By governing the endocytic fate of internalized receptors, Odin may provide a layer of regulation that enables cells to contend with receptor cell densities and ligand concentration gradients that are physiologically and pathologically highly variable.</p></div

Effect of Odin expression on EGF induced EGFR degradation.

<p>A–B, Whole cell lysates from HEK293 cells (Cont), or this cell type stably expressing ectopic Odin (Odin), a control non-silencing shRNA (shCont), or an Odin-directed shRNA (shOdin) were prepared after treatment with EGF (10 ng/ml, 37°C) for the indicated durations, and then western blotted with the indicated antibodies. The results shown are representative of three experiments.</p

The effect of Odin expression on EGFR trafficking.

<p>A–C, EGFR endocytosis after 30 min EGF treatment. Serum-deprived Cont, Odin, ShCont and ShOdin cells were incubated on ice with EGF (50 ng/ml) for 30 min, washed free of unbound ligand, and then provided pre-warmed ligand-free medium at 37°C for 30 min. The first column shows bright field or direct green fluorescence (which identifies lentivirus-infected cells). The cells were fixed and stained with EGFR antibodies (red, second column) and antibodies to EEAI (panel A), Rab11 (panel B), or LAMP1 (panel C) (blue, third column), wherein pink indicates co-localization (Merge, fourth column). Insets show an additional 3-fold magnification of the boxed area. Scale bars are 5 µm.</p

Phosphorylation of the adaptor-like protein Odin in ligand-stimulated and stress-activated cells.

<p>A, Schematic drawing of the domain structure of Odin (not to scale) showing amino acid numbers of the indicated features; adapted from Pandey et al. (36). HEK293 cells that stably express EGFR-Flag were subjected to anti-Odin IP of endogenous Odin (B) after treatment with EGF at 37°C or 4°C. The IPs and whole cell lysates were analyzed by Western blot for phosphotyrosine (pY), Odin, and phosphorylated (pERK) or total ERK1/2 proteins (ERK). C, Western blot analysis of anti-myc IPs from the same cells after transient transfection with an Odin-myc expression vector, and treatment with EGF (100 ng/ml) for the indicated durations, and with TGFα (50 ng/ml), TNFα (100 ng/ml), and anisomycin (Aniso; 10 µM) for 15 min. Results shown are representative of three independent experiments.</p

The effect of Odin expression on EGFR trafficking.

<p>EGFR co-localization with EEA1, Rab11, and LAMP1, after the indicated incubation time with EGF was quantified by using Volocity software (Perkin Elmer). Co-localization coefficients (mean ±SEM, n = 10) represent pixel overlap between EGFR and EEA1, Rab11, or LAMP1. The coefficient ranges from 0 to 1, with 0 corresponding to non-overlapping images and 1 corresponding to 100% overlap. Asterisks above bars indicate a statistically significant difference from control (Odin v. Cont; shOdin v. shCont); and asterisks above connecting lines indicates the significance of the difference between the connected bars: * <i>p</i><0.05; ** <i>p</i><0.01.</p

A Novel Mechanism for SUMO System Control: Regulated Ulp1 Nucleolar Sequestration ▿

The small ubiquitin-related modifiers (SUMOs) are evolutionarily conserved polypeptides that are covalently conjugated to protein targets to modulate their subcellular localization, half-life, or activity. Steady-state SUMO conjugation levels increase in response to many different types of environmental stresses, but how the SUMO system is regulated in response to these insults is not well understood. Here, we characterize a novel mode of SUMO system control: in response to elevated alcohol levels, the Saccharomyces cerevisiae SUMO protease Ulp1 is disengaged from its usual location at the nuclear pore complex (NPC) and sequestered in the nucleolus. We further show that the Ulp1 region previously demonstrated to interact with the karyopherins Kap95 and Kap60 (amino acids 150 to 340) is necessary and sufficient for nucleolar targeting and that enforced sequestration of Ulp1 in the nucleolus significantly increases steady-state SUMO conjugate levels, even in the absence of alcohol. We have thus characterized a novel mechanism of SUMO system control in which the balance between SUMO-conjugating and -deconjugating activities at the NPC is altered in response to stress via relocalization of a SUMO-deconjugating enzyme