Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)–based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.

ABSTRACTaKlotho is a multifunctional protein highly expressed in the kidney. Soluble aKlotho is released through cleavage of the extracellular domain from membrane aKlotho by secretases to function as an endocrine/paracrine substance. The role of the kidney in circulating aKlotho production and handling is incompletely understood, however. Here, we found higher aKlotho concentration in suprarenal compared with infrarenal inferior vena cava in both rats and humans. In rats, serum aKlotho concentration dropped precipitously after bilateral nephrectomy or upon treatment with inhibitors of aKlotho extracellular domain shedding. Furthermore, the serum half-life of exogenous aKlotho in anephric rats was four-to five-fold longer than that in normal rats, and exogenously injected labeled recombinant aKlotho was detected in the kidney and in urine of rats. Both in vivo (micropuncture) and in vitro (proximal tubule cell line) studies showed that aKlotho traffics from the basal to the apical side of the proximal tubule via transcytosis. Thus, we conclude that the kidney has dual roles in aKlotho homeostasis, producing and releasing aKlotho into the circulation and clearing aKlotho from the blood into the urinary lumen.

We present a yeast chemical-genomics approach designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds. We developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes. The MoBY-ORF resource has numerous genetic and chemical-genetic applications, but here we focus on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays. The complementation cloning was validated by mutation detection using whole-genome yeast tiling microarrays, which identified unique polymorphisms associated with a drug-resistant mutant. We used the MoBY-ORF library to identify the genetic basis of several drug-resistant mutants and in this analysis discovered a new class of sterol-binding compounds.

Internalization of diverse transmembrane cargos from the plasma membrane requires a similarly diverse array of specialized adaptors, yet only a few adaptors have been characterized. We report the identification of the muniscin family of endocytic adaptors that is conserved from yeast to human beings. Solving the structures of yeast muniscin domains confirmed the unique combination of an N‐terminal domain homologous to the crescent‐shaped membrane‐tubulating EFC/F‐BAR domains and a C‐terminal domain homologous to cargo‐binding μ homology domains (μHDs). In vitro and in vivo assays confirmed membrane‐tubulation activity for muniscin EFC/F‐BAR domains. The μHD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. The transmembrane protein Mid2, earlier implicated in polarized Rho1 signalling, was identified as a cargo of the yeast adaptor protein. These and other data suggest a model in which the muniscins provide a combined adaptor/membrane‐tubulation activity that is important for regulating endocytosis.