EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Statistical protein quantification and significance analysis in label-free LC-MS experiments with complex designs.

Background: Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is widely used for quantitative proteomic investigations. The typical output of such studies is a list of identified and quantified peptides. The biological and clinical interest is, however, usually focused on quantitative conclusions at the protein level.

Hst3 is regulated by Mec1-dependent proteolysis and controls the S phase checkpoint and sister chromatid cohesion by deacetylating histone H3 at lysine 56.

The SIR2 homologues HST3 and HST4 have been implicated in maintenance of genome integrity in the yeast Saccharomyces cerevisiae. We find that Hst3 has NAD-dependent histone deacetylase activity in vitro and that it functions during S phase to deacetylate the core domain of histone H3 at lysine 56 (H3K56). In response to genotoxic stress, Hst3 undergoes rapid Mec1-dependent phosphorylation and is targeted for ubiquitin-mediated proteolysis, thus providing a mechanism for the previously observed checkpoint-dependent accumulation of Ac-H3K56 at sites of DNA damage.

Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.

The ATP binding cassette (ABC) transporters are important in human health and disease and represent the largest family of transmembrane proteins; however, their highly hydrophobic nature complicates the use of standard biochemical approaches to identify interacting proteins. Here, we report the development of a modified version of the split-ubiquitin membrane yeast two-hybrid (MYTH) technology using genomically integrated "bait" constructs, hence the designation iMYTH. We used iMYTH in a library-screening format and identified six potential interacting partners of the yeast ABC transporter Ycf1p.

Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins.

Various modifications of the conventional yeast two-hybrid system have played an essential role in confirming or detecting protein-protein interactions among nuclear and cytoplasmic proteins. These approaches have permitted the identification of novel interaction partners, as well as provided hints as to their function. However, membrane proteins, such as receptor tyrosine kinases, G protein-coupled receptors, membrane-bound phosphatases, and transporters, which represent important classes of signaling molecules, are difficult to study using classical protein interaction assays because of their hydrophobic nature.

The split-ubiquitin membrane-based yeast two-hybrid system.

Protein-protein interactions are essential in almost all biological processes, extending from the formation of cellular macromolecular structures and enzymatic complexes to the regulation of signal transduction pathways. It is assumed that approximately one-third of all proteins in eukaryotic cells are membrane associated. Because of their hydrophobic nature, the analysis of membrane-protein interactions is difficult to be studied in a conventional two-hybrid assay.

Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein.

Secretory proteins are translocated across the endoplasmic reticulum (ER) membrane through a channel formed by three proteins, namely Sec61p, Sbh1p, and Sss1p (Johnson, A. E., and van Waes, M.

Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system.

Analysis of membrane protein interactions is difficult because of the hydrophobic nature of these proteins, which often renders conventional biochemical and genetic assays fruitless. This is a substantial problem because proteins that are integral or associated with membranes represent approximately one-third of all proteins in a typical eukaryotic cell. We have shown previously that the modified split-ubiquitin system can be used as a genetic assay for the in vivo detection of interactions between the two characterized yeast transmembrane proteins, Ost1p and Wbp1p.

The post-genomic era of interactive proteomics: facts and perspectives.

The availability of completed genome sequences of several eukaryotic and prokaryotic species has shifted the focus towards the identification and characterization of all gene products that are expressed in a given organism. In order to cope with the huge amounts of data that have been provided by large-scale sequencing projects, high-throughout methodologies also need to be applied in the emerging field of proteomics. In this review, we discuss methods that have been recently developed in order to characterize protein interactions and their functional relevance on a large scale.

Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays.

Interaction of two separately expressed halves of sucrose transporter SUT1 was detected by an optimized split-ubiquitin system. The halves reconstitute sucrose transport activity at the plasma membrane with affinities similar to the intact protein. The halves do not function independently, and an intact central loop is not required for membrane insertion, plasma membrane targeting, and transport.