Genome-wide conditional degron libraries for functional genomics.

Functional genomics with libraries of knockout alleles is limited to non-essential genes and convoluted by the potential accumulation of suppressor mutations in knockout backgrounds, which can lead to erroneous functional annotations. To address these limitations, we constructed genome-wide libraries of conditional alleles based on the auxin-inducible degron (AID) system for inducible degradation of AID-tagged proteins in the budding yeast Saccharomyces cerevisiae. First, we determined that N-terminal tagging is at least twice as likely to inadvertently impair protein function across the proteome.

Multiple quality control mechanisms monitor yeast chitin synthase folding in the endoplasmic reticulum.

The chitin synthase Chs3 is a multipass membrane protein whose trafficking is tightly controlled. Accordingly, its exit from the endoplasmic reticulum (ER) depends on several complementary mechanisms that ensure its correct folding. Despite its potential failure on its exit, Chs3 is very stable in this compartment, which suggests its poor recognition by ER quality control mechanisms such as endoplasmic reticulum-associated degradation (ERAD).

Genetic requirements for repair of lesions caused by single genomic ribonucleotides in S phase.

Single ribonucleoside monophosphates (rNMPs) are transiently present in eukaryotic genomes. The RNase H2-dependent ribonucleotide excision repair (RER) pathway ensures error-free rNMP removal. In some pathological conditions, rNMP removal is impaired.

High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein Timers.

Tandem fluorescent protein timers (tFTs) are versatile reporters of protein dynamics. A tFT consists of two fluorescent proteins with different maturation kinetics and provides a ratiometric readout of protein age, which can be exploited to follow intracellular trafficking, inheritance and turnover of tFT-tagged proteins. Here, we detail a protocol for high-throughput analysis of protein turnover with tFTs in yeast using fluorescence measurements of ordered colony arrays.