Mary Lui

RAFT1: A mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs

The immunosuppressants rapamycin and FK506 bind to the same intracellular protein, the immunophilin FKBP12. The FKB12-FK506 complex interacts with and inhibits the Ca(2+)-activated protein phosphatase calcineurin. The target of the FKBP12-rapamycin complex has not yet been identified. We report that a protein complex containing 245 kDa and 35 kDa components, designated rapamycin and FKBP12 targets 1 and 2 (RAFT1 and RAFT2), interacts with FKBP12 in a rapamycin-dependent manner. Sequences (330 amino acids total) of tryptic peptides derived from the 245 kDa RAFT1 reveal striking homologies to the yeast TOR gene products, which were originally identified by mutations that confer rapamycin resistance in yeast. A RAFT1 cDNA was obtained and found to encode a 289 kDa protein (2549 amino acids) that is 43% and 39% identical to TOR2 and TOR1, respectively. We propose that RAFT1 is the direct target of FKBP12-rapamycin and a mammalian homolog of the TOR proteins.

The Gβγ Sensitivity of a PI3K Is Dependent upon a Tightly Associated Adaptor, p101

Two highly similar, PtdIns(4,5)P2-selective, G beta gamma-activated PI3Ks were purified from pig neutrophil cytosol. Both were heterodimers, were composed of a 101 kDa protein and either a 120 kDa or a 117 kDa catalytic subunit, and were activated greater than 100-fold by G beta gammas. Peptide sequence-based oligonucleotide probes were used to clone cDNAs for the p120 and p101 species. The cDNA of p120 is highly related to p110 gamma, while the cDNA of p101 is not substantially related to anything in current databases. The proteins were expressed in and purified from insect and mammalian cells. They bound tightly to one another, both in vivo and in vitro, and in so doing, p101 amplified the effect of G beta gammas on the PI3K activity of p120 from less than 2-fold to greater than 100-fold.

Identification of New Mediator Subunits in the RNA Polymerase II Holoenzyme from Saccharomyces cerevisiae

Mediator was isolated from yeast on the basis of its requirement for transcriptional activation in a fully defined system. We have now identified three new members of mediator in the low molecular mass range by peptide sequence determination. These are the products of the NUT2, CSE2, and MED11 genes. The product of the NUT1 gene is evidently a component of mediator as well. NUT1 and NUT2 were earlier identified as negative regulators of the HO promoter, whereas mutations in CSE2 affect chromosome segregation.MED11 is a previously uncharacterized gene. The existence of these proteins in the mediator complex was verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.

MALDI-TOF Mass Spectrometry in the Protein Biochemistry Lab: From Characterization of Cell Cycle Regulators to the Quest for Novel Antibiotics

For decades, direct covalent analysis was the only way to get the full primary structure of a protein, a tedious task involving several digests and exhaustive analysis [1]. More recently, this process has been accelerated by the use of limited sequence information to assist in cloning of the corresponding genes [2, 3]. Cloned DNA can be readily analyzed and the entire protein primary structure deduced. With a large number of genes sequenced and repositories rapidly expanding [4, 5], partial protein sequences have also allowed, with increasing frequency, matching biological function (or regulation) to a specific database entry [6, 7, 8]. The focus of protein sequencing has therefore again shifted to questions of protein associations in the cell. After discovery of one or more components of a functional complex (in signal transduction, cell cycle and differentiation, and vesicle targeting, among other processes), the question typically arises with which other proteins they might interact [9, 10, 11]. Since many of the targets are only available in minute quantities, it is imperative that analytical studies be carried out at the highest levels of sensitivity. Such pioneering applications challenge scientists and engineers, and drive technical developments. Frequently, multi-analytical approaches are taken to maximize accuracy. In this regard, it has been long foretold that mass spectrometry would attain a very prominent role in the protein chemistry lab.

Protein Micro-Characterization by Mass Spectrometry: Sample Handling and Data Flow

Mass spectrometry (MS) occupies center position in most current protein identification schemes. “Mass fingerprinting” techniques rely on composite mass patterns of proteolytic fragments, or dissociation products thereof, to query databases [1–6]. Two issues, however, need to be addressed at the present time. Are we ready for high throughput analyses in a reliable, efficient and timely manner, and to apply these tools towards rationally selected, meaningful research problems?