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K Lehman

Publications and source records attributed to K Lehman.

9 recordsLinked to original sources
PubMed2001

Importance of homodimerization for the in vivo function of yeast RNA triphosphatase.

Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus. The active site of Cet1 resides within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the crystallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys(330) and Val(331) and a cross-dimer hydrogen bond of Asp(280) with the backbone amide of Gln(329). The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe(272) and Leu(273). Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity but sedimented as monomers. These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase. We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo.

Acid Anhydride Hydrolases↗
PubMed2000

Characterization of the mRNA capping apparatus of Candida albicans.

The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520- amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p). The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals. Hence, the triphosphatase is an attractive antifungal target. Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520. We find that CaCet1p function in vivo requires the segment from residues 202 to 256 immediately flanking the catalytic domain from 257 to 520. Genetic suppression data implicate the essential flanking segment in the binding of CaCet1p to the fungal guanylyltransferase. Deletion analysis of the Candida guanylyltransferase demarcates an N-terminal domain, Cgt1(1-387)p, that suffices for catalytic activity in vitro and for cell growth. An even smaller domain, Cgt1(1-367)p, suffices for binding to the guanylyltransferase docking site on yeast RNA triphosphatase. Deletion analysis of the cap methyltransferase identifies a C-terminal domain, Ccm1(137-474)p, as being sufficient for cap methyltransferase function in vivo and in vitro. Ccm1(137-474)p binds in vitro to synthetic peptides comprising the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II. Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat. We show that the entire three-component Saccharomyces cerevisiae capping apparatus can be replaced by C. albicans enzymes. Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes.

Acid Anhydride Hydrolases↗
PubMed2000

Characterization of Candida albicans RNA triphosphatase and mutational analysis of its active site.

The RNA triphosphatase component (CaCet1p) of the mRNA capping apparatus of the pathogenic fungus Candida albicans differs mechanistically and structurally from the RNA triphosphatase of mammals. Hence, CaCet1p is an attractive antifungal target. Here we identify a C-terminal catalytic domain of CaCet1p from residue 257 to 520 and characterize a manganese-dependent and cobalt-dependent NTPase activity intrinsic to CaCet1p. The NTPase can be exploited to screen in vitro for inhibitors. The amino acids that comprise the active site of CaCet1p were identified by alanine-scanning mutagenesis, which was guided by the crystal structure of the homologous RNA triphosphatase from Saccharomyces cerevisiae (Cet1p). Thirteen residues required for the phosphohydrolase activity of CaCet1p (Glu287, Glu289, Asp363, Arg379, Lys396, Glu420, Arg441, Lys443, Arg445, Asp458, Glu472, Glu474 and Glu476) are located within the hydrophilic interior of an eight-strand beta barrel of Cet1p. Each of the eight strands contributes at least one essential amino acid. The essential CaCet1p residues include all of the side chains that coordinate manganese and sulfate (i.e., gamma phosphate) in the Cet1p product complex. These results suggest that the active site structure and catalytic mechanism are conserved among fungal RNA triphosphatases.

Acid Anhydride Hydrolases↗
PubMed1999

An essential surface motif (WAQKW) of yeast RNA triphosphatase mediates formation of the mRNA capping enzyme complex with RNA guanylyltransferase.

Saccharomyces cerevisiae RNA triphosphatase (Cet1p) and RNA guanylyltransferase (Ceg1p) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Cet1p binding to Ceg1p stimulates the guanylyltransferase activity of Ceg1p. Here we localize the guanylyltransferase-binding and guanylyltransferase-stimulation functions of Cet1p to a 21-amino acid segment from residues 239 to 259. The guanylyltransferase-binding domain is located on the protein surface, as gauged by protease sensitivity, and is conserved in the Candida albicans RNA triphosphatase CaCet1p. Alanine-cluster mutations of a WAQKW motif within this segment abolish guanylyltransferase-binding in vitro and Cet1p function in vivo, but do not affect the triphosphatase activity of Cet1p. Proteolytic footprinting experiments provide physical evidence that Cet1p interacts with the C-terminal domain of Ceg1p. Trypsin-sensitive sites of Ceg1p that are shielded from proteolysis when Ceg1p is bound to Cet1p are located between nucleotidyl transferase motifs V and VI.

Acid Anhydride Hydrolases↗
PubMed1999

A conserved domain of yeast RNA triphosphatase flanking the catalytic core regulates self-association and interaction with the guanylyltransferase component of the mRNA capping apparatus.

The 549-amino acid yeast RNA triphosphatase Cet1p catalyzes the first step in mRNA cap formation. Cet1p consists of three domains as follows: (i) a 230-amino acid N-terminal segment that is dispensable for catalysis in vitro and for Cet1p function in vivo; (ii) a protease-sensitive segment from residues 230 to 275 that is dispensable for catalysis but essential for Cet1p function in vivo; and (iii) a catalytic domain from residues 275 to 539. Sedimentation analysis indicates that purified Cet1(231-549)p is a homodimer. Cet1(231-549)p binds in vitro to the yeast RNA guanylyltransferase Ceg1p to form a 7.1 S complex that we surmise to be a trimer consisting of two molecules of Cet1(231-549)p and one molecule of Ceg1p. The more extensively truncated protein Cet1(276-549)p, which cannot support cell growth, sediments as a monomer and does not interact with Ceg1p. An intermediate deletion protein Cet1(246-549)p, which supports cell growth only when overexpressed, sediments principally as a discrete salt-stable 11.5 S homo-oligomeric complex. These data implicate the segment of Ceg1p from residues 230 to 275 in regulating self-association and in binding to Ceg1p. Genetic data support the existence of a Ceg1p-binding domain flanking the catalytic domain of Cet1p, to wit: (i) the ts growth phenotype of 2mu CET1(246-549) is suppressed by overexpression of Ceg1p; (ii) a ts alanine cluster mutation CET1(201-549)/K250A-W251A is suppressed by overexpression of Ceg1p; and (iii) 15 other cet-ts alleles with missense changes mapping elsewhere in the protein are not suppressed by Ceg1p overexpression. Finally, we show that the in vivo function of Cet1(275-549)p is completely restored by fusion to the guanylyltransferase domain of the mouse capping enzyme. We hypothesize that the need for Ceg1p binding by yeast RNA triphosphatase can by bypassed when the triphosphatase catalytic domain is delivered to the RNA polymerase II elongation complex by linkage in cis to the mammalian guanylyltransferase.

Acid Anhydride Hydrolases↗
PubMed1999

Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9.

We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

Actins↗
PubMed1999

Nursing and medical student teaming for service learning in partnership with the community: an emerging holistic model for interdisciplinary education and practice.

To meet the health needs of communities today, health professionals need to be trained in working with persons from various cultural backgrounds, practicing disease prevention and health promotion in community-based settings, and working in teams with other professionals. The article focuses on interdisciplinary teaming for education and practice. In this model, medical and nursing students partner with communities to plan and deliver health promotion education programs and activities. Four service learning projects providing collaborative teaming opportunities as part of the Health Professions Schools in Service to the Nation Program are described. Interdisciplinary service learning has benefits for the community, students, and faculty and will prepare nurses and physicians to have a positive impact on care through future interdisciplinary collaboration in community-based settings.

Community Health Nursing↗
PubMed1997

Reducing costs through case management.

Three studies over a 3-year period compare nursing case management (NCM) outcomes--resources, service costs and NCM costs. NCM continues to improve quality of patient care in a cost-effective way.

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