Regulation of mannan synthesis in Saccharomyces cerevisiae

Harrington, Charles R. (1980) Regulation of mannan synthesis in Saccharomyces cerevisiae. PhD thesis, University of Glasgow.

Abstract

Although a great deal is known about the chemical structure and enzymatic mechanisms involved in the assembly of yeast mannoproteins, little is known about the regulation of mannan biosynthesis. The aim of this study, therefore, was to investigate the control mechanisms involved. Two approaches were used. Firstly, the potential role of low molecular-weight effectors of mannosyltransferases was investigated. Secondly, the possible existence of an activation-inactivation mechanism of control, analogous to that which operates during chitin synthesis was explored. Mannosyltransferase activity in sphaeroplast lysates and washed membranes prepared from Saccharomyces cerevisiae X2180 was measured by following the incorporation of [14C]cjmannose from GDP-[14C]nBnnose into material precipitable with cold 0.3M perchloric acid. Mannosyl- transferase activity was optimal at low enzyme protein concentrations (l mg/ml), 50°C and neutral pH values; with 7.5 mM MnCl2; it did not require exogenous mannoprotein acceptors. Mannosyltransferase activity from both sphaeroplast lysate and washed membrane preparations was not markedly affected by sugars, nucleoside monophosphates, TJDP, TTDP-N-acetyl glucosamine, ADP-, CDP-, TDP- and UBP-glucose. UDP- and ABP-mannose, GDP-glucose, GDP and GTP all inhibited mannosyltransferase activity from both preparations while activity was enhanced by ABP and ATP. UTP enhanced activity from the washed membrane fraction but inhibited that from the sphaeroplast lysate. The stimulatory effects of ATP and UTP were dependent upon the exact method of sphaeroplast lysate preparation. These results suggest that UTP and ATP levels may function in the control of mannosyl transferase activity. GDP-glucose acted as a potent competitive inhibitor of mannosyltransferase activity. When enzyme activity was assayed at high concentrations of sphaeroplast lysate protein (10 mg/ml), with 7.5 MnCl2, a severe inhibition was observed. This inhibition could be relieved by preincubation of the sphaeroplast lysate or by omission of MnCl2 from assay mixtures. The addition of EDTA or monovalent cations removed inhibition in the presence of Mn2+. No similar inhibition was observed when a washed membrane fraction was substituted for sphaeroplast lysate as the source of mannosyltransferase activity. The supernatant fraction obtained by centrifuging sphaeroplast lysate at 100,000 x g, when added to assay mixtures containing either sphaeroplast lysate preincubated at or washed membrane fraction, also caused inhibition of enzyme activity. This inhibition required 7.5 inM MnCl2, was destroyed by heating the supernatant fraction at 60°C for 10 min, or by trypsin or pepsin treatment at and was gradually lost when the supernatant fraction was stored at 4°C. The supernatant fraction caused inhibition of synthesis of both (3-eliminable oligosaccharides and polysaccharide chains of mannan. These results indicate the existence of a protein inhibitor of mannan synthesis whose inhibitory activity in sphaeroplast lysates may be modulated by preincubation at low temperature or by varying the available MnCl2, concentration. These findings indicate that activation-inactivation processes exist in the regulation of mannan as well as chitin biosynthesis. A model, proposed by Farkas (1979). for yeast cell-wall formation is discussed in which the results of this study on mannan synthesis have been integrated.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Dr. L.J. Douglas.
• Keywords: Biochemistry.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Supervisor, not known
• Date of Award: 1980
• Depositing User: Enlighten Team
• Unique ID: glathesis:1980-72319
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 May 2019 15:12
• Last Modified: 27 Jun 2022 14:16
• Thesis DOI: 10.5525/gla.thesis.72319
• URI: https://theses.gla.ac.uk/id/eprint/72319