An Investigation into the Effects of the E5 Family of Transforming Proteins on the Vacuolar Proton-Translocating ATPase

Ashby, Andrew Daniel Mark (2000) An Investigation into the Effects of the E5 Family of Transforming Proteins on the Vacuolar Proton-Translocating ATPase. PhD thesis, University of Glasgow.

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There are a number of viruses known to be causative agents of cancer (zur Hausen, 1991b). Most of these viruses act through expression of proteins that interfere with the action of cellular proteins involved in regulating cell division. This thesis examines the effects of various forms of E5 proteins, including a related protein from a retrovirus (HTLV-1) using Saccharomyces cerevisiae as a model organism. Yeast was chosen as it can grow in the absence of V-ATPase activity, unlike higher eukaryotic cells, and much is known about its V-ATPase. As S. cerevisiae is not the host organism of papillomaviruses and HTLV-1, the binding of the E5 proteins to yeast form of ductin was first examined. In an in vitro assay using co-translation and immunoprecipitation, all E5 proteins were found to bind to the S. cerevisiae form of ductin. When E5 proteins were expressed in S. cerevisiae using a vector known to give high expression of membrane proteins, all E5 proteins were detected in the vacuoles, a membrane which is the main site for the V-ATPase. Monitoring growth of the transformed yeast strains showed the expression of E5 proteins was not cytotoxic. The effects of the E5 transforming proteins were next tested on the activity of the S. cerevisiae V-ATPase. This study was extended to include a mutant form of ductin in which a key glutamic acid residue (E137) had been changed to glycine and is known to act as a dominant negative (Hughes et al, 1996). This mutant prevented growth at pH 7.5 and in high extracellular Calcium, both restrictive growth conditions that require V-ATPase activity. However, none of the E5 proteins, despite binding to ductin, had any effect. The enzyme was then examined more directly by kinetic analysis of ATP hydrolysis. None of the E5 proteins perturbed the activity of the V-ATPase as measured by Km or Vmax. Replacing glucose with galactose/raffinose in the growth medium of yeast reduces the V-ATPases activity (Kane, 1995). Repeating these experiments showed an initial complete loss of V-ATPase activity but the activity recovered to approximately 40% of the original value. This compromised system was therefore used to examine any effects of E5 transforming proteins. Whilst the dominant negative form of ductin again disrupted growth in the restrictive conditions, none of the E5 proteins had any effect. The apparent lack of effect on V-ATPase activity by E5 proteins was further investigated by examining the locations of the V-ATPase and E5 proteins after separation by size fractionation on glycerol gradients after ultracentrifugation. This showed that a representative of the E5 transforming proteins, HPV-16 E5, did not co-purify with the V-ATPase indicating it became detached from the active enzyme. The separation explains why E5 transforming proteins, although capable of binding to yeast ductin, do not disturb V-ATPase function. A partially disabled V-ATPase was used to discover if the E5 proteins could disturb activity. This form contained the Norway lobster (Nephrops norvegicus) ductin tagged with hexa-histidine tail at the C-terminus, known to markedly elevate the Km for ATP (Harrison et al, 1994). Again, the dominant negative form of ductin ablated growth at high pH, but none of the E5 proteins had any effect. Therefore, the E5 transforming proteins do not affect V-ATPase activity and it seems likely that their transforming activity is not occurring through the V-ATPase as has been thought. A scheme is proposed which takes into account recent findings on the related enzyme F-ATPase by which the binding to ductin is part of the mechanism of targeting E5 transforming proteins from the Endoplasmic Reticulum to other cellular compartments. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Malcolm Finbow
Keywords: Molecular biology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-74933
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:07
Last Modified: 27 Sep 2019 15:07

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