The O-GlcNAc transferase (OGT) is a unique nuclear and cytosolic glycosyltransferase that contains multiple tetratricopeptide repeats. We have begun to characterize the mechanisms regulating OGT using a combination of deletion analysis and kinetic studies. Here we show that the p110 subunit of the enzyme forms both homoand heterotrimers that appear to have different binding affinities for UDP-GlcNAc. The multimerization domain of OGT lies within the tetratricopeptide repeat domain and is not necessary for activity. Kinetic analyses of the full-length trimer and the truncated monomer forms of OGT suggest that both forms function through a random bi-bi kinetic mechanism. Both the monomer and trimer have similar specific activities and similar K m values for peptide substrates. However, they differ in their binding affinities for UDP-GlcNAc, indicating that subunit interactions affect enzyme activity. The findings that recombinant OGT has three distinct K m values for UDP-GlcNAc and that UDP-GlcNAc concentrations modulates the affinity of OGT for peptides suggest that OGT is exquisitely regulated by the levels of UDP-GlcNAc within the nucleus and cytoplasm.O-GlcNAc is an abundant intracellular posttranslational modification consisting of a single N-acetylglucosamine O-linked to serine/threonine residues. Unlike other carbohydrate modifications, O-GlcNAc is not further modified and is found almost exclusively in the nucleus and cytoplasm. Since it was first described in lymphocytes (1) O-GlcNAc has been found on an ever increasing number of proteins, including RNA polymerase II and its transcription factors, nuclear pore proteins, tumor suppressor proteins, intermediate filaments, viral proteins, and oncoproteins (reviewed in Refs. 2-5). O-GlcNAc is an abundant and dynamic modification exhibiting properties more like phosphorylation than typical N-and O-linked glycosylation (2, 3). The O-GlcNAc modification (termed O-GlcNAcylation) has been suggested to play a direct role in regulating a number of cellular functions including protein synthesis (6, 7), neurofilament assembly (8), and transcription (9 -11). In our laboratory, we have purified and characterized both a UDP-Nacetylglucosamine:peptide N-acetylglucosaminyl-transferase (O-GlcNAc transferase) (12) specific for the attachment of OGlcNAc to proteins and a soluble N-acetyl--D-glucosaminidase (O-GlcNAcase) (13) specific for the removal of O-GlcNAc from proteins. These enzymes may work together to regulate the attachment and removal of O-GlcNAc in response to cellular signals in much the same way that kinases and phosphatases regulate protein phosphorylation.Although significant progress has been made in our understanding of the distribution of O-GlcNAc in the cell, little is known about how the attachment of O-GlcNAc to proteins is regulated. The problem is significant because numerous proteins are O-GlcNAcylated, many at more then one site. A further complexity is added by the lack of a canonical consensus site for the attachment of O-GlcNAc to proteins (4, 5)...
O-Linked N-acetylglucosamine (O-GlcNAc) glycosylation is a dynamic modification of eukaryotic nuclear and cytosolic proteins analogous to protein phosphorylation. We have cloned and characterized a novel gene for an O-GlcNAc transferase (OGT) that shares no sequence homology or structural similarities with other glycosyltransferases. The OGT gene is highly conserved (up to 80% identity) in all eukaryotes examined. Unlike previously described glycosyltransferases, OGT is localized to the cytosol and nucleus. The OGT protein contains multiple tandem repeats of the tetratricopeptide repeat motif. The presence of tetratricopeptide repeats, which can mediate protein-protein interactions, suggests that OGT may be regulated by protein interactions that are independent of the enzyme's catalytic site. The OGT is also modified by tyrosine phosphorylation, indicating that tyrosine kinase signal transduction cascades may play a role in modulating OGT activity.Unlike other forms of protein glycosylation, serine (threonine)-O-linked N-acetylglucosamine (O-GlcNAc) is found primarily in the cytoplasm and nucleus, and is not modified or elongated to more complex structures (1, 2). Since it was first described in 1984 (3), the O-GlcNAc modification (termed OGlcNAcylation) has been found on a myriad of eukaryotic nuclear and cytosolic proteins, including RNA polymerase II and its transcription factors, nuclear pore proteins, viral proteins, cytoskeletal proteins, tumor suppressor proteins, and oncoproteins (reviewed in Refs. 1 and 2).Direct evidence is rapidly accumulating in support of the role of O-GlcNAcylation as a regulatory modification. O-GlcNAc appears to be as abundant as phosphorylation, and several of the known sites of attachment are similar to those used by proline-directed kinases (4, 5). O-GlcNAcylation is a dynamic modification exhibiting properties more like phosphorylation than typical O-and N-linked glycosylation (1, 2). The turnover rate of the O-GlcNAc moiety on cytokeratins (6) and the small heat shock protein ␣B-crystallin (7) is much higher than the turnover rate of the protein. O-GlcNAcylation also has been shown to regulate a number of cellular functions. For example recent studies have shown the following. 1) O-GlcNAcylation modulates the DNA binding activity of the p53 tumor suppressor (8). 2) O-GlcNAcylation of p67 regulates protein synthesis by controlling the phosphorylation state of the elongation initiation factor 2 (eIF-2␣) (9, 10). 3) O-GlcNAcylation of the head domain of neurofilament-H appears to regulate neurofilament assembly (11). 4) The O-GlcNAc and phosphate modifications of the RNA polymerase II COOH-terminal domain are reciprocal and are likely to regulate transcription (12, 13). 5) O-GlcNAc has a reciprocal relationship with phosphorylation at the site on the c-Myc protein, which has been implicated in modulating its oncogenic activity (14).Consistent with the dynamic nature of O-GlcNAcylation, both a UDP-N-acetylglucosamine:peptide N-acetylglucosaminyl-transferase (O-GlcNAc transferase...
In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal‐binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide‐containing phytochelatin‐cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP‐binding cassette)‐type transport proteins which includes the mammalian P‐glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1‐expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue‐specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants.
Dictyostelium discoideum has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions. The relatively small ( approximately 34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We now describe a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying four separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers.
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