Yeast Irc6p is a novel type of conserved clathrin coat accessory factor related to small G proteins.

Clathrin coat accessory proteins play key roles in transport mediated by clathrin-coated vesicles. Yeast Irc6p and the related mammalian p34 are putative clathrin accessory proteins that interact with clathrin adaptor complexes. We present evidence that Irc6p functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p.

Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek's microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology.

Genome-wide analysis of AP-3-dependent protein transport in yeast.

The evolutionarily conserved adaptor protein-3 (AP-3) complex mediates cargo-selective transport to lysosomes and lysosome-related organelles. To identify proteins that function in AP-3-mediated transport, we performed a genome-wide screen in Saccharomyces cerevisiae for defects in the vacuolar maturation of alkaline phosphatase (ALP), a cargo of the AP-3 pathway. Forty-nine gene deletion strains were identified that accumulated precursor ALP, many with established defects in vacuolar protein transport.

High-throughput protein extraction and immunoblotting analysis in Saccharomyces cerevisiae.

A variety of Saccharomyces cerevisiae strain libraries allow for systematic analysis of strains bearing gene deletions, repressible genes, overexpressed genes, or modified genes on a genome-wide scale. Here we introduce a method for culturing yeast strains in 96-well format to achieve log-phase growth and a high-throughput technique for generating whole-cell protein extracts from these cultures using sodium dodecyl sulfate and heat lysis. We subsequently describe a procedure to analyze these whole-cell extracts by immunoblotting for alkaline phosphatase and carboxypeptidase yscS to identify strains with defects in protein transport pathways or protein glycosylation.

Structural and functional insights into the regulation of Arabidopsis AGC VIIIa kinases.

The AGCVIIIa kinases of Arabidopsis are members of the eukaryotic PKA, PKG, and PKC group of regulatory kinases. One AGCVIIIa kinase, PINOID (PID), plays a fundamental role in the asymmetrical localization of membrane proteins during polar auxin transport. The remaining 16 AGCVIIIa genes have not been associated with single mutant phenotypes, suggesting that the corresponding kinases function redundantly.

Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast.

Discovering target and off-target effects of specific compounds is critical to drug discovery and development. We generated a compendium of "chemical-genetic interaction" profiles by testing the collection of viable yeast haploid deletion mutants for hypersensitivity to 82 compounds and natural product extracts. To cluster compounds with a similar mode-of-action and to reveal insights into the cellular pathways and proteins affected, we applied both a hierarchical clustering and a factorgram method, which allows a gene or compound to be associated with more than one group.