The Arabidopsis LEAFY COTYLEDON1 (LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box-binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.
We carried out a large-scale screen to identify interactions between integral membrane proteins of Saccharomyces cerevisiae by using a modified split-ubiquitin technique. Among 705 proteins annotated as integral membrane, we identified 1,985 putative interactions involving 536 proteins. To ascribe confidence levels to the interactions, we used a support vector machine algorithm to classify interactions based on the assay results and protein data derived from the literature. Previously identified and computationally supported interactions were used to train the support vector machine, which identified 131 interactions of highest confidence, 209 of the next highest confidence, 468 of the next highest, and the remaining 1,085 of low confidence. This study provides numerous putative interactions among a class of proteins that have been difficult to analyze on a high-throughput basis by other approaches. The results identify potential previously undescribed components of established biological processes and roles for integral membrane proteins of ascribed functions.Saccharomyces cerevisiae ͉ split-ubiquitin ͉ support vector machine S ystematic studies of protein interactions in yeast have provided insights into the functions of many of the proteins encoded by this single-celled eukaryote. However, the roles of many integral membrane proteins remain poorly understood. Biochemical purifications require detergents to isolate proteins away from lipid molecules, and the large-scale nature of the affinity precipitation͞mass spectrometry projects (1, 2) precluded adjusting the detergents for individual integral membrane proteins. Two-hybrid assays (3, 4) require that the two proteins localize to the nucleus; integral membrane proteins, targeted to an aqueous nuclear environment, may aggregate or misfold.To increase the representation of integral membrane proteins in the protein-protein interaction network of Saccharomyces cerevisiae, we examined pair-wise interactions among 705 integral membrane proteins by the split-ubiquitin membrane yeast two-hybrid system (5). This modified form of the split-ubiquitin assay (6-8) is one of several hybrid protein approaches that detect interactions occurring at membranes. The split-ubiquitin membrane yeast two-hybrid system allows direct identification of yeast transformants that encode a pair of interacting proteins by use of a transcriptional reporter.Analyses of previous large-scale interaction data sets revealed significant numbers of false negatives and false positives (9). False negatives may represent interactions unsuitable for detection by a particular technique and, thus, may not be easily remedied. False positives can potentially be identified by a failure to be validated through additional experiments. However, the large-scale nature of this study and the difficulties associated with biochemical analysis of integral membrane proteins preclude confirmation of these results by alternative experimental approaches. Therefore, we used a learning algorithm, the support vector machi...
Random mutagenesis methods only partially cover the mutational space, and are constrained by DNA synthesis length limitations. Here, we demonstrate PALS, a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.
Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activityenhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.NMR | phage display | ubiquitin E3 ligase | protein stability | deep mutational scanning C ovalent modification of proteins by ubiquitin (Ub) has an impact on nearly all eukaryotic cell biology. The attachment of Ub is accomplished by three enzymes: an E1 Ub-activating enzyme, an E2 Ub-conjugating enzyme, and an E3 Ub ligase (1). In the final stage of the pathway, the E3 ligase facilitates transfer from a Ub-loaded E2 (termed an E2∼Ub conjugate) onto a substrate lysine. Minimally, functional E3s contain an E2-binding domain and a substrate-recognition domain, enabling them to bind an E2∼Ub and a substrate simultaneously. The majority of E3s harbor either a RING (really interesting new gene) domain or a related U-box domain to bind cognate E2s. RING-type E3s enhance transfer of Ub directly from an E2's active site to a substrate lysine without an intermediate transfer step of Ub to the E3 itself, as occurs with homology to E6AP carboxyl-terminus (HECT)-type ligases. In addition to providing proximity between the Ub attached to the active site of an E2 and a substrate amino group, RING-type E3s activate E2∼Ub conjugates allosterically (2-11). Allosteric activation relies on promotion of catalytically active "closed" conformations of an E2∼Ub conjugate that presumably arrange the E2 active site thioester for access and attack by an incoming nucleophile. Thus, two processes contribute to the rate enhancement of Ub transfer by RING-type E3s: (i) proximity (and, in some cases, orientational) effects and (ii) promotion of reactive states of the E2∼Ub. Recent studies (8-10) have shown that minimal RING-type domains that lack a substrate-binding activity (and therefore cannot provide a proximity enhancement) are able to enhance the intrinsic reactivity of E2∼Ub species, demonstrating that the two sources of rate enhancemen...
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