Matthias Meurer

Tandem fluorescent protein timers for in vivo analysis of protein dynamics

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.

Protein quality control at the inner nuclear membrane

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

One library to make them all: streamlining the creation of yeast libraries via a SWAp-Tag strategy

The yeast Saccharomyces cerevisiae is ideal for systematic studies relying on collections of modified strains (libraries). Despite the significance of yeast libraries and the immense variety of available tags and regulatory elements, only a few such libraries exist, as their construction is extremely expensive and laborious. To overcome these limitations, we developed a SWAp-Tag (SWAT) method that enables one parental library to be modified easily and efficiently to give rise to an endless variety of libraries of choice. To showcase the versatility of the SWAT approach, we constructed and investigated a library of ∼1,800 strains carrying SWAT-GFP modules at the amino termini of endomembrane proteins and then used it to create two new libraries (mCherry and seamless GFP). Our work demonstrates how the SWAT method allows fast and effortless creation of yeast libraries, opening the door to new ways of systematically studying cell biology.

Seamless Gene Tagging by Endonuclease-Driven Homologous Recombination

Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.

Artificial tethering to nuclear pores promotes partitioning of extrachromosomal DNA during yeast asymmetric cell division

SUMMARY Asymmetric cell division in unicellular organisms enables sequestration of senescence factors to specific subpopulations. Accumulation of autonomously replicating sequence (ARS) plasmids, which frequently emerge from recombination within the highly repetitive ribosomal DNA locus, is linked to limited replicative life span of Saccharomyces cerevisiae cells [1]. During budding yeast cell division, ARS plasmids are retained in the ageing mother cell, such that only 1 out of 10 plasmids enters the rejuvenated bud [2]. Binding of ARS plasmids to nuclear structures retained in the mother cell was speculated to explain asymmetric plasmid segregation [2]. Association with nuclear pore complexes (NPCs) was proposed to underlie retention of ARS plasmids in the mother cell [3]. However, the role of NPCs in segregation of ARS plasmids is unclear, as NPCs are partitioned between mother and bud nuclei during mitosis [4,5]. Here we analyzed how segregation of ARS plasmids is influenced by their interaction with NPCs. We found that artificial tethering to NPCs promotes transport of ARS plasmids into the bud. Moreover, our experiments provide support for the notion that interaction with ARS plasmids does not affect movement of NPCs into the bud. We conclude that binding to NPCs cannot by itself contribute to asymmetric segregation of ARS plasmids.

Protein Abundance Control by Non-coding Antisense Transcription

Summary Stable unannotated transcripts (SUTs), some of which overlap protein-coding genes in antisense direction, are a class of non-coding RNAs. While case studies have reported important regulatory roles for several of such RNAs, their general impact on protein abundance regulation of the overlapping gene is not known. To test this, we employed seamless gene manipulation to repress antisense SUTs of 162 yeast genes by using a unidirectional transcriptional terminator and a GFP tag. We found that the mere presence of antisense SUTs was not sufficient to influence protein abundance, that observed effects of antisense SUTs correlated with sense transcript start site overlap, and that the effects were generally weak and led to reduced protein levels. Antisense regulated genes showed increased H3K4 di- and trimethylation and had slightly lower than expected noise levels. Our results suggest that the functionality of antisense RNAs has gene and condition-specific components.

Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

Tandem fluorescent protein timers (tFTs)—fusions of red and green fluorescent proteins—facilitate studies of intracellular protein dynamics. Various green fluorescent proteins are resistant to proteasomal degradation, and incomplete degradation affects tFT behavior. Simple guidelines are given for the design of new tFTs.

Quantification of cytosolic interactions identifies Ede1 oligomers as key organizers of endocytosis

Clathrin‐mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein–protein interactions between up to 60 different proteins. However, little is known about the spatio‐temporal regulation of these interactions. Using fluorescence (cross)‐correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self‐interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein–protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo.

Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

An improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, is introduced, called hmKeima8.5. Its increased intracellular brightness and long Stokes shift (~180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Here we demonstrate its utility in intracellular fluorescence correlation spectroscopy applications.

PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast

Here, we report on a novel PCR targeting-based strategy called ‘PCR duplication’ that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning.