Mutants yeast

The Rieske protein in mitochondrial bci complexes is assembled when the protein is incorporated into the complex. The Rieske protein is encoded in the nucleus and synthesized in the cytosol with a mitochondrial targeting presequence, which is required to direct the apoprotein to the mitochondrial matrix. The C-terminus is then targeted back to the outside of the inner mitochondrial membrane where the Rieske cluster is assembled. In addition, the presequence is removed and the protein is processed to its mature size after the protein is inserted into the bci complex. In mammals, the presequence is cleaved in a single step by the core proteins 1 and 2, which are related to the general mitochondrial matrix processing protease (MPP) a and (3 subunits the bovine heart presequence is retained as a 8.0 kDa subunit of the complex (42, 107). In Saccharomyces cerevis-iae, processing occurs in two steps Initially, the yeast MPP removes 22 amino acid residues to convert the precursor to the intermediate form, and then the mitochondrial intermediate protease (MIP) removes 8 residues after the intermediate form is in the bci complex (47). Cleavage by MIP is independent of the assembly of the Rieske cluster Conversion of the intermediate to the mature form was observed in a yeast mutant that did not assemble any Rieske cluster (35). However, in most mutants where the assembly of the Rieske cluster is prevented, the amount of Rieske protein is drastically reduced, most likely because of instability (35, 44). 

Experimental approaches that have afforded major insights to the processes described in this chapter include (1) use of yeast mutants (2) application of recombinant DNA techniques (eg, mutating or eliminating particular sequences in proteins, or fusing new sequences onto them and (3) development of in vitro... 

Nurse There are yeast mutants that capture the astral microtubules and orient the nucleus. It may be interesting to look at homologues of these. 

Simon I don t think it is known. Some years ago researchers rounded up the usual suspects from yeast, complemented yeast mutants and checked for the expression patterns of factors such as the cyclins and Cdcs in plant meristems. They saw some spotty expression patterns in the meristems, but there were no clear domains of down-regulation of cell divisions, for example. 

Allen, J. Broadhurst, H. M. Heald, J. K. Rowland, J. J. Oliver, S. G Kell, D. B. High-throughput classification of yeast mutants for functional genomics using metabolic footprinting. Nat. Biotechnol. 2003, 21, 692-696. 

H., Huber, R., et al. Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants. J. Biol. Chem. 1998, 273, 25637-25646. 

A successful tool in the early studies of metabolic pathways was blocking the pathway at some specific point. This could be done by the use of either mutants or inhibitors. Schekman et al have isolated a number of yeast mutants with blocks in their secretion pathway (Schekman, 1982). It is not yet known which proteins these mutations affect, but this is clearly a most promising approach for identifying those components involved in transport. In animal cells there are no cellular mutants with blocks in the intracellular transport of protein from the ER to the cell surface. There are, however, genetic diseases which affect the routing of lysosomal enzymes to the lysosomes (Neufeld et al, 1975 Sly and Fischer, 1982). For viruses it has been possible to isolate temperature-sensitive mutants in which a mutation in the viral glycoprotein arrests... 

Cell cycle is one of the physiological processes in which the role of uhiquitin—proteasome-mediated proteolysis is well established. With the advent of yeast mutants that interfered with various phases of the cell cycle, cyclin-dependent kinases (Cdks) were found to have a critical role in regulating the cell cycle. Typically, Cdks activated hy regulatory proteins are known as cyclins. Different Cdk—cyclin complexes are formed at specific stages of the cell cycle such as the S-phase (in which DNA synthesis occurs) and the metaphase. The transition from metaphase to anaphase depends on degradation of cyclins. " " Systematic biochemical studies showed that cyclins were substrates for the uhiquitin—proteasome pathway (Table 5). 

Li, Y., T. Kane, C. Tipper, P. Spatrick, and D. D. Jeimess. Yeast mutants affecting possible quality control of plasma membrane proteins. Mol Cell Biol. 19 3588-99.1999. 

Yeast does not synthesise cycloartenol or triterpenes, and so approaches to clone plant OSCs by complementation in S. cerevisiae are not feasible because of the lack of appropriate mutants. However, LS-deficient yeast mutants accumulate high levels of 2,3-oxidosqualene, favouring the synthesis of novel cycfisation products generated by heterologous expression of OSCs. The absence of lano-sterol also facilitates analysis of the reaction products. Corey and co-workers isolated a cDNA encoding Arabidopsis thaliana CS by transforming a plant cDNA expression library into such a yeast mutant and screening protein preparations derived from pools of transformants for the ability to synthesise cycloartenol by TLC [39]. 

Yaffe MP, Ohta S, Schatz G (1985) A yeast mutant temperature-sensitive for mitochondrial assembly is deficient in a mitochondrial protease activity that cleaves imported precursor polypeptides. EMBO J 4 2069-2074... 

A series of specific inhibitors of trimming reactions, some of whose structures are shown in Fig. 20-7, has provided important insights 253-255 Use of these inhibitors, together with immunochemical methods and study of yeast mutants,250 252 256 is enabling us to learn many details of glycoprotein biosynthesis. 

M Lancien, P Gadal, M Hodges. Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase evidence for a heteromeric structure by the complementation of yeast mutants. Plant J 16 325-333, 1998. 

A second desaturase-encoding cDNA was isolated from this species and found to encode a 352 amino acid protein that is orthologous to the H. zea A9 desaturase HzPGDs2 based on its 83 percent sequence identity to the latter vs only 63-64 percent identity to the TnFBDs and HZFBDs desaturases. When this cDNA, designated Pocto-Z9, was functionally expressed in the olel yeast mutant it... 

A 34 kDa protein (p34) plays an important function in the control of the cell cycle in all eukaryotes. It was first identified as the product of the cdc 2/cdc 28 gene in yeast mutants which caused cells to be arrested at a commitment point in G1 (Murray, 1981). However, anti-p34 antibodies injected into cells do not affect DNA synthesis but block cells in mitosis (Riabowol et al., 1989) and p34 is believed to function both at the onset of S-phase and at mitosis. 

In certain contemporary organisms, for instance, bacteria and fungi, 3-phospho-D-glyceroyl-phosphate polyphosphate phosphotransferase activity was found (Kulaev and Bobyk, 1971 Kulaev et al., 1971). The phosphate was transferred from 1,3-diphosphoglyceric acid, not to ADP to form ATP, as one could expect from the Meyerhof-Embden-Parnas scheme, but directly to PolyP. This fossil reaction was most expressed in an adenine deficient yeast mutant under cell adaptation to ATP depletion. 

---