Biomedical subjects

Safia Thaminy

Publications and source records attributed to Safia Thaminy.

6 recordsLinked to original sources

PubMed2005

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. Here, we describe a genetic system that allows the identification of integral membrane-interacting proteins. This so-called "split-ubiquitin membrane-based yeast two-hybrid assay" involves fusing the halves of ubiquitin to two interacting proteins, at least one of which is membrane bound. Upon interaction of these two proteins, the halves of ubiquitin are brought together, and the transcription factor that is fused to a membrane protein of interest is cleaved and released. The free transcription factor then enters the nucleus and activates transcription of reporter genes. We also describe how this technology is used to screen complementary DNA libraries to identify novel binding partners of a membrane protein of interest.

Amino Acid Substitution↗

PubMed2004

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. We described here a new genetic method for in vivo detection of membrane-protein interactions in the budding yeast Saccharomyces cerevisiae. The system uses the split-ubiquitin approach based on the detection of the in vivo processing of a reconstituted split ubiquitin. On interaction of X and Y proteins, ubiquitin reconstitution occurs and leads to the proteolytic cleavage and subsequent release of a transcription factor that triggers the activation of a reporter system enabling easy detection. In this manner, and in contrast to the conventional yeast-two hybrid system in which interactions occur in the nucleus, the membrane-based yeast two-hybrid system represents an in vivo system that detects interactions between membrane proteins in their natural environment.

Amino Acid Sequence↗

PubMed2003

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. A. (1999) Annu. Rev. Cell Dev. Biol. 15, 799-842). Sec61p and Sss1p are essential for translocation (Esnault, Y., Blondel, M. O., Deshaies, R. J., Schekman, R., and Kepes, F. (1993) EMBO J. 12, 4083-4093). Sec61p is a polytopic membrane protein that lines the protein translocation channel. The role of Sss1p is unknown. During import into the ER through the Sec61p channel, many proteins are N-glycosylated before translocation is completed. In addition, both the Sec61 channel and oligosaccharyl transferase (OST) copurify with ribosomes from rough ER, suggesting that OST is located in close proximity to the Sec61 channel (Gorlich, D., Prehn, S., Hartmann, E., Kalies, K.-U., and Rapoport, T. A. (1992) Cell 71, 489-503 and Wang, L., and Dobberstein, B. (1999) FEBS Lett. 457, 316-322). Here, we demonstrate a direct interaction between Sss1p and a subunit of OST, Wbp1p, using the split-ubiquitin system and co-immunoprecipitation. We generated mutants in the cytoplasmic domain of Sss1p that disturb the interaction with OST and are viable but display a translocation defect specific for proteins with glycosylation acceptor sites. Our data suggest that Sss1p coordinates translocation across the ER membrane and N-linked glycosylation of secretory proteins.

Amino Acid Sequence↗

PubMed2003

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. This so-called split-ubiquitin membrane yeast two-hybrid (YTH) system uses the split-ubiquitin approach in which reconstitution of two ubiquitin halves is mediated by a protein-protein interaction. Here we converted the split-ubiquitin membrane YTH system into a generally applicable in vivo screening approach to identify interacting partners of a particular mammalian transmembrane protein. We have demonstrated the effectiveness of this approach by using the mammalian ErbB3 receptor as bait and have identified three previously unknown ErbB3-interacting proteins. In addition, we have confirmed one of the newly found interactions between ErbB3 and the membrane-associated RGS4 protein by coimmunoprecipitating the two proteins from human cells. We expect the split-ubiquitin membrane YTH technology to be valuable for the identification of potential interacting partners of integral membrane proteins from many model organisms.

Amino Acid Sequence↗

PubMed2002

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. We then focus on those methodologies that are suitable for the identification and characterization of protein-protein interactions, namely the yeast two-hybrid system and related methods. Several recent studies have demonstrated the power of automated approaches involving the yeast two-hybrid system in building so-called "interaction networks", which hold the promise of identifying the entirety of all interactions that take place between proteins expressed in a certain cell type or organism. We compare the yeast two-hybrid system with several other screening methods that have been developed to investigate interactions between proteins that are not amenable to conventional yeast two-hybrid screenings, such as transcriptional activators and integral membrane proteins. The eventual adaptation of such methods to a high-throughput format and their use in combination with automated yeast two-hybrid screenings will help in elucidating protein-protein interactions on a scale that would have been unthinkable just a few years ago.

Genome, Fungal↗

PubMed2002

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. Under native conditions, the halves associate into higher molecular mass complexes. Furthermore, the N-terminal half of the low-affinity SUT2 interacts functionally with the C-terminal half of SUT1. Since the N terminus of SUT2 determines affinity for sucrose, the reconstituted chimera has lower affinity than SUT1. The split-ubiquitin system efficiently detects intramolecular interactions in membrane proteins, and can be used to dissect transporter structure.

Amino Acid Sequence↗