Skip to main content
Article thumbnail
Location of Repository

The characterisation of Plc1 : a phospholipase C enzyme identified in the fission yeast Schizosaccharomyces pombe

By Robert Ian Brind

Abstract

The plc1 gene product (Plc1) of the fission yeast Schizosaccharomycespombe\ud (Sz. pombe) encodes a phosphoinositide-specific phospholipase C (PLC) and\ud most closely resembles the δ class of the PLC isozymes. PLC hydrolyses\ud phosphatidylinositol 4,5-bisphosphate (PIP2) producing two second messengers,\ud inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). The work in this\ud thesis is concerned with aspects of PLC signalling in Sz. pombe cells.\ud Sz. pombe cells lacking p1c1 (Δplc1) display a distinct phenotype. Δplc1 cells are\ud viable they grow slowly at 29'C (and below) on rich medium, but are unable to\ud grow under stressful conditions such as on minimal medium or at 37'C. Loss of\ud Plc1 function has been shown to cause missegregation of chromosomes and\ud enlarged cells with aberrant morphology (Fankhauser et al. 1995). Expression of\ud Plc1 in Δplc1 cells complemented for the loss of Plc1 and resulted in a wild-type\ud Sz. pombe phenotype.\ud Described in this thesis are the characterisation experiments carried out on Plc1.\ud Extracts prepared from wild-type Sz. pombe cells have undetectable Plc1 activity,\ud so an assay was designed, to measure Plc1 activity in vitro. The in vitro assay\ud and expression of Plc1 in Δplc1 cells formed the basis of a set of experiments\ud that helped identify a possible regulatory domain and roles for Plc1 in Sz. pombe\ud cells. Mutant forms of Plc1 were assayed in vitro to determine their level of\ud activity and were then expressed in Δplc1 cells to see if they were able to\ud complement for the loss of Plc1 activity.\ud Temperature sensitive mutants of Plc1 were constructed. This illustrated what\ud happened to a Sz. pombe cell with an active Plc1 at 23'C but when incubated at\ud 37'C Plc1 activity is switched off. This demonstrated that cells lacking Plc1\ud activity could no longer divide and defects in cell wall structure began to appear.\ud Active site mutants of Plc1 were produced to identify whether InSP3 or DAG are\ud important second messengers in Sz. pombe cells. The Plc1 mutants were unable\ud to hydrolyse PIP2 but could hydrolyse phosphatidylinositol (PI). These mutants\ud hydrolyse PI producing DAG and InsP and were able to rescue the Δplc1\ud phenotype. This suggests that DAG production may play an important role in\ud regulating stress response pathways whereas InsP is recycled to produce\ud phosphatidylinositols.\ud Yeast PLCs have an extended N-terminal domain, which is not seen in any other\ud eukaryotic PLCs. To investigate the role of this N-terminal domain, mutants of\ud Plc1 and PLC-6 were constructed. They demonstrated that without the N-terminus\ud these mutants were active in vitro but were unable to complement for\ud the loss of Plc1 activity in Δplc1 cells. This indicates that the N-terminal domain\ud may play an important regulatory role

Topics: QR
OAI identifier: oai:wrap.warwick.ac.uk:4018

Suggested articles

Citations

  1. (1987). Evidence that inositol I -phosphatein brain of lithium-treated rats resulted mainly from phosphatidylinositol metabolism.
  2. (1995). Regulation of inositol lipid-specific phospholipase C6 by changes in Ca" ion concentrations.
  3. (1996). Evidence that the Gh protein is a signal mediator from the aI -adrenoreceptor to a phospholipase C.
  4. SH3 domains direct cellular localisation of signalling molecules.
  5. (1993). Adaptor self-aggregation, adaptor-receptor recognition and binding of oc-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP-2) components of clatherin coated pits.
  6. (1995). Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis Of PIP2- doi
  7. (1995). Pheromone procedures in fission yeast. doi
  8. (1993). A single C2 domain from synaptotagmin I is sufficient for high affinity Ca 2+ /phospholipid binding.
  9. (1990). Phosphoinositides as regulators in membrane traffic.
  10. (1992). The mammalian ultraviolet response is triggered by activation of src tyrosine kinases. doi
  11. (1994). ARF: a key regulatory switch in membrane traffic and organelle structure. doi
  12. (1995). Crystal structure of mammalian phosphoinositde-specific phospholipase C6.
  13. (1995). Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. doi
  14. (1998). An essential function of a pho sphatidylino sito I -specific phospholipase C is relieved by inhibition of a cyclin-dependent protein kinase in the yeast Saccharomyces cerevisiae.
  15. (1993). Phosphatidylinositol-specific phospholipase C of Bacillus cereus: cloning, sequencing and relationship to other phospholipases.
  16. (1992). Inositol phosphate biochemistry. doi
  17. (1994). Gh: A GTP-binding protein with transglutarninase activity and receptor signalling function.
  18. (1992). Presence of SH2 domains of phospholipase C71 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor.
  19. (1995). Mutational analysis of a putative polyphosphoinositide binding site in phospholipase C-P2. doi
  20. (1993). Regulation of purified subtypes of phosphatidylinositol-specific phospholipase Q by G-protein oc and P7 subunits.
  21. (1995). Receptor mediated protein sorting to the vacuole in yeast: Roles for a protein kinase and lipid kinase and GTP binding prOteins. doi
  22. (1998). Phosphoinositde-specific phospholipase C 81 activity toward micellar substrates, inositol 1,2-cyclic phosphate, and other water-soluble substrates: A sequential mechanism and allosteric activation. doi
  23. (1995). The putative phosphoinositide-specific phospholipase C gene, PLC], of the yeast Saccharomyces cerevisiae is important for cell growth. doi

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.