The characterisation of Plc1 : a phospholipase C enzyme identified in the fission yeast Schizosaccharomyces pombe
Brind, Robert Ian (2000) The characterisation of Plc1 : a phospholipase C enzyme identified in the fission yeast Schizosaccharomyces pombe. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b1370977~S15
The plc1 gene product (Plc1) of the fission yeast Schizosaccharomycespombe
(Sz. pombe) encodes a phosphoinositide-specific phospholipase C (PLC) and
most closely resembles the δ class of the PLC isozymes. PLC hydrolyses
phosphatidylinositol 4,5-bisphosphate (PIP2) producing two second messengers,
inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). The work in this
thesis is concerned with aspects of PLC signalling in Sz. pombe cells.
Sz. pombe cells lacking p1c1 (Δplc1) display a distinct phenotype. Δplc1 cells are
viable they grow slowly at 29'C (and below) on rich medium, but are unable to
grow under stressful conditions such as on minimal medium or at 37'C. Loss of
Plc1 function has been shown to cause missegregation of chromosomes and
enlarged cells with aberrant morphology (Fankhauser et al. 1995). Expression of
Plc1 in Δplc1 cells complemented for the loss of Plc1 and resulted in a wild-type
Sz. pombe phenotype.
Described in this thesis are the characterisation experiments carried out on Plc1.
Extracts prepared from wild-type Sz. pombe cells have undetectable Plc1 activity,
so an assay was designed, to measure Plc1 activity in vitro. The in vitro assay
and expression of Plc1 in Δplc1 cells formed the basis of a set of experiments
that helped identify a possible regulatory domain and roles for Plc1 in Sz. pombe
cells. Mutant forms of Plc1 were assayed in vitro to determine their level of
activity and were then expressed in Δplc1 cells to see if they were able to
complement for the loss of Plc1 activity.
Temperature sensitive mutants of Plc1 were constructed. This illustrated what
happened to a Sz. pombe cell with an active Plc1 at 23'C but when incubated at
37'C Plc1 activity is switched off. This demonstrated that cells lacking Plc1
activity could no longer divide and defects in cell wall structure began to appear.
Active site mutants of Plc1 were produced to identify whether InSP3 or DAG are
important second messengers in Sz. pombe cells. The Plc1 mutants were unable
to hydrolyse PIP2 but could hydrolyse phosphatidylinositol (PI). These mutants
hydrolyse PI producing DAG and InsP and were able to rescue the Δplc1
phenotype. This suggests that DAG production may play an important role in
regulating stress response pathways whereas InsP is recycled to produce
Yeast PLCs have an extended N-terminal domain, which is not seen in any other
eukaryotic PLCs. To investigate the role of this N-terminal domain, mutants of
Plc1 and PLC-6 were constructed. They demonstrated that without the N-terminus
these mutants were active in vitro but were unable to complement for
the loss of Plc1 activity in Δplc1 cells. This indicates that the N-terminal domain
may play an important regulatory role.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QR Microbiology|
|Library of Congress Subject Headings (LCSH):||Schizosaccharomyces pombe, Phospholipase C, Second messengers (Biochemistry)|
|Official Date:||February 2000|
|Institution:||University of Warwick|
|Theses Department:||Department of Biological Sciences|
|Sponsors:||Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)|
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