Daniel Lockshon

A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae

Two large-scale yeast two-hybrid screens were undertaken to identify protein–protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.

A role for recombination junctions in the segregation of mitochondrial DNA in yeast

In S. cerevisiae, mitochondrial DNA (mtDNA) molecules, in spite of their high copy number, segregate as if there were a small number of heritable units. The rapid segregation of mitochondrial genomes can be analyzed using mtDNA deletion variants. These small, amplified genomes segregate preferentially from mixed zygotes relative to wild-type mtDNA. This segregation advantage is abolished by mutations in a gene, MGT1, that encodes a recombination junction-resolving enzyme. We show here that resolvase deficiency causes a larger proportion of molecules to be linked together by recombination junctions, resulting in the aggregation of mtDNA into a small number of cytological structures. This change in mtDNA structure can account for the increased mitotic loss of mtDNA and the altered pattern of mtDNA segregation from zygotes. We propose that the level of unresolved recombination junctions influences the number of heritable units of mtDNA.

The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function

The peroxisome, sole site of β-oxidation in Saccharomyces cerevisiae, is known to be required for optimal growth in the presence of fatty acid. Screening of the haploid yeast deletion collection identified ∼130 genes, 23 encoding peroxisomal proteins, necessary for normal growth on oleic acid. Oleate slightly enhances growth of wild-type yeast and inhibits growth of all strains identified by the screen. Nonperoxisomal processes, among them chromatin modification by H2AZ, Pol II mediator function, and cell-wall-associated activities, also prevent oleate toxicity. The most oleate-inhibited strains lack Sap190, a putative adaptor for the PP2A-type protein phosphatase Sit4 (which is also required for normal growth on oleate) and Ilm1, a protein of unknown function. Palmitoleate, the other main unsaturated fatty acid of Saccharomyces, fails to inhibit growth of the sap190Δ, sit4Δ, and ilm1Δ strains. Data that suggest that oleate inhibition of the growth of a peroxisomal mutant is due to an increase in plasma membrane porosity are presented. We propose that yeast deficient in peroxisomal and other functions are sensitive to oleate perhaps because of an inability to effectively control the fatty acid composition of membrane phospholipids.

The yeast two-hybrid system: prospects for protein linkage maps

Every cell contains a set of machines capable of carrying out processes such as replication or transcription, responding to the outside environment, generation of usable energy or directing the traffic flow of macromolecules. These machines are like intricate three-dimensional jigsaw puzzles, forming arrays of interlocking protein components that assemble and disassemble over time and in response to complex signals. Understanding cellularfunction is advanced by knowledge of each assembly, which requires an inventory of its parts and a blueprint that details how these parts fit together. A simple genetic assay in yeast, the two-hybrid system1f2, detects protein-protein interactions and can be used to analyse many such assemblies. In principle, it may be possible to employ this assay to identify most of the components in these protein complexes, and the resulting networks of interactions are whatwe refer to as a ‘protein linkage map’. Traditionally, assemblies of proteins have been analysed by using two complementary approaches. In the well-known analogy to understanding how a car runs, biochemists disassemble the engine, transmission and body, characterize all the pieces, and attempt to rebuild a working vehicle; geneticists, by contrast, break single components, turn the key and try to determine what effect the single missing part has on the car’s operation. These strategies have proved spectacularly successful in deciphering many of the essential processes of a cell, and undoubtedly will continue to be fruitful. However, the advent of genome sequencing projects of organisms ranging in complexity from mycoplasma to man (reviewed in Ref. 3) has ushered in the arrival of thousands of new proteins to analyse, and there is difficulty in applying such labour-intensive traditional procedures to so many proteins. This onslaught of genomic information calls for the development of efficient experimental strategies to gain insight into the organization and function of the encoded proteins.

Sir2 deletion prevents lifespan extension in 32 long-lived mutants

Activation of Sir2 orthologs is proposed to increase lifespan downstream of dietary restriction. Here, we describe an examination of the effect of 32 different lifespan‐extending mutations and four methods of DR on replicative lifespan (RLS) in the short‐lived sir2Δ yeast strain. In every case, deletion of SIR2 prevented RLS extension; however, RLS extension was restored when both SIR2 and FOB1 were deleted in several cases, demonstrating that SIR2 is not directly required for RLS extension. These findings indicate that suppression of the sir2Δ lifespan defect is a rare phenotype among longevity interventions and suggest that sir2Δ cells senesce rapidly by a mechanism distinct from that of wild‐type cells. They also demonstrate that failure to observe lifespan extension in a short‐lived background, such as cells or animals lacking sirtuins, should be interpreted with caution.

Rho Signaling Participates in Membrane Fluidity Homeostasis

Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is differentially influenced by its two principal unsaturated fatty acids, oleic and palmitoleic acid. Strains deficient in the core components of the cell wall integrity (CWI) pathway, a MAP kinase pathway dependent on both Pkc1 (yeasts sole protein kinase C) and Rho1 (the yeast RhoA-like small GTPase), were among those inhibited by palmitoleate yet stimulated by oleate. A single GEF (Tus1) and a single GAP (Sac7) of Rho1 were also identified, neither of which participate in the CWI pathway. In contrast, key components of the CWI pathway, such as Rom2, Bem2 and Rlm1, failed to influence fatty acid sensitivity. The differential influence of palmitoleate and oleate on growth of key mutants correlated with changes in membrane fluidity measured by fluorescence anisotropy of TMA-DPH, a plasma membrane-bound dye. This work provides the first evidence for the existence of a signaling pathway that enables eukaryotic cells to control membrane fluidity, a requirement for division, differentiation and environmental adaptation.