Here, we have identified ptn1p, an S. pombe homologue of mammalian PTEN. The ptn1p phosphatase domain is 38% homologous to the human PTEN phosphatase domain, and all of the residues essential for PIP phosphatase activity are conserved. Furthermore, recombinant ptn1p dephosphorylates PI(3,4,5)P3 and cells lacking ptn1 show markedly increased levels of PI(3,4)P2 and PI(3,4,5)P3. Based on these findings, we reach the surprising conclusion that S. pombe has a true PTEN orthologue that regulates the levels of PI(3,4)P2 and PI(3,4,5)P3.
The discovery of ptn1
led us to examine the biosynthetic pathway for PI(3,4,5)P3
synthesis. We discovered a novel pathway that originates with synthesis of PI(3)P by vps34p, followed by the conversion of PI(3)P to PI(3,4,5)P3
. Its3p, the S. pombe
orthologue of mammalian type I PIP 5-kinases, converts PI(3)P into PI(3,4)P2
, as has been shown to occur for mammalian type I PIP 5-kinases (Zhang et al., 1997
; Tolias et al., 1998
). The enzyme that catalyzes the last step in the synthesis of PI(3,4,5)P3
has not been identified, but by analogy with the mammalian pathway, may also be its3p. The observation that wild-type cells have undetectable or very low levels of PI(3,4)P2
indicates that, as for mammalian cells, these lipids are tightly regulated in fission yeast. This regulation may occur at the level of synthesis and/or degradation of these lipids. Here we show that the ptn1p has an important role in maintaining the low levels of PI(3,4)P2
in S. pombe.
Understanding the spatial and temporal regulation of PI(3,4)P2
synthesis are important questions for future studies.
Ptn1p, like PI(3)P (Gillooly et al., 2000
) and vps34p (Stack et al., 1995
), was found associated with vesicular structures, and ptn1
Δ cells show irregularly shaped vacuoles and are more readily lysed by osmotic stress. However, we also observed ptn1p associated with the septa of dividing cells. Hence, as in mammalian cells, PI(3,4,5)P3
) in S. pombe
likely has multiple functions, regulating different processes in different regions of the cell.
The mechanism by which this lipid affects cell function in fission yeast remains to be determined. One can imagine that, as for mammalian cells, PI(3,4,5)P3
) may function to recruit target proteins to specific subcellular locations via binding to protein modules. The S. pombe
genome includes 21 putative PH domains (Wood et al., 2002
), which in mammalian cells bind to PI phosphates and mediate many of the downstream effects. Our investigation of the phosphoinositide binding specificity of S. pombe
PH domains revealed that the GFP-11E3.11C PH domain has distinct subcellular distributions in wild-type and ptn1
Δ cells, suggesting that it is regulated by PI(3,4)P2
. However, by the filter binding assay the 11E3.11C PH domain is not specific for PI(3,4)P2
. There are several possible explanations. First, there may be experimental complications, relating to incomplete folding of in vitro translated PH domains, thereby, compromising PH domain specificity. In addition, binding of PH domains to filters is an excellent method for surveying phosphoinositide specificity, but binding of PH domains to undiluted phosphoinositides on a filter is sometimes less selective than in biological membranes (Snyder et al., 2001
). Second, specific binding of S. pombe
PH domains to membranes might require interactions with both lipid and protein targets. Indeed, some S. cerevisiae
PH domains require multiple interactions for membrane binding (Yu et al., 2004
). Third, localization of the 11E3.11C PH domain in ptn1
Δ cells may be due to a higher affinity for PI(3,4,5)P3
, as has been observed for the ARNO PH domain (Venkateswarlu et al., 1998
; Cullen and Chardin, 2000
). The 11E3.11C predicted protein is a homologue of the ARNO/cytohesin/Grp family and like these mammalian proteins, has an Arf GDP/GTP exchange domain and PH domain. Hence, PI(3,4,5)P3
in lower eukaryotes may act through a PH domain (domains) that binds multiple phosphoinositides, and PI(3,4,5)P3
-specific PH domains may have evolved in more complex species.
In summary, the results presented here indicate that a pathway for the synthesis of PI(3,4,5)P3 from PI(3)P existed in yeast before the evolution of class I PI 3-kinases in higher eukaryotes, indicating a more ancient function for this important signaling molecule.