The vital role that inositides and their components play in the regulation of cell events – The synthesis and metabolism of Phosphotidylinositol(3,5)bisphosphate

Published: 2019/12/10 Number of words: 3026

1.1 Phosphotidylinositol(3,5)bisphosphate: Synthesis and

In the year 1850, Joseph Scherer discovered inositol and determined its empirical formula by its isolation from Liebig’s extract of meat. Inositides consist of phosphotidyl-inositol (PtdIns), polyphosphoinositides (PPIns) which are a phosphorylated derivative of PtdIns and inositol polyphosphate(IP). These membrane and cellular components of inositides have been found to play a vital role in the regulation of cellular events (Kangzhen Dong, 2010).

PtdIns, a member of eukaryotic membrane phospholipids, play an important role in phosphorylation of the inositol group in one or more than one place, giving rise to various PtdIns (Corvera et al., 1999). These phosphoinositides are found near the cytoplasmic face. They act as a substrate for various enzymes which convert them into secondary messengers, including PtdIns (3,5) bisphosphate Corvera et al., 1999). These enzymes include phosphoinositol kinase, phospholipase C and phospholipase D (Robinson et al., 1998). The inter-conversions of PPIns are controlled by various lipid phospholipases and kinases. Usually these enzymes will phosphor late or dephosphorylate at a particular hydroxyl group on the inositol ring (Dong., 2010).

Phosphotidyl inositol (3,5) bisphosphate (PtdIns (3,5)P2) is a newly discovered member of PPIns and plays a role in membrane trafficking (Michell et al., 2006), cytoskeletal rearrangement and responding to extracellular environmental changes (Strahl et al., 2007). It is also involved in packing proteins into multivesicular bodies after being ubiquitinated, for growth at high temperatures and for vacuole acidification (Dove et al., 2004).

In 1987, Auger et al. postulated the existence of PtdIns(3,5)P2 but its presence was not confirmed until 1997, when it was isolated and identified in a mouse’s fibroblast and in Saccharomyces cerevisiae (Whiteford et al., 1997; Dove et al., 1997). Its presence was also reported in Schizosaccharomyces pombe and in plant cells, suggesting that it exists in all eukaryotes (Dove et al., 1997).

The presence of PtdIns kinase has been observed in various eukaryotic organisms, including yeast, insects and vertebrates. These kinases have been classified into three types: type I/type III PtdIns kinase, type II PtdIns kinase and PtdIns phosphokinase. PtdIns 4 kinase, which is a type II kinase, was the first to be isolated from various mammalian sources and described in detail (Strahl et al., 2004). Later, it was realised that these PtdIns kinase catalyse a reaction on a specific inositol ring (Balla et al., 2006) and were classified according to the product they produce: PtdIns 3-kinase, PtdIns 4-kinase and PtdInsP kinase (see the review of Strahl and Thorner, 2007).

PtdIns are converted to PtdIns 3 phosphate by the action of type III PPIn

3-kinase and then to PtdIns (3,5)P2 by the action of PIPKIII (Michell et al.,2006). In Saccharomyces cerevisiae, VPS34p is responsible for the conversion of PtdIns to PtdIns 3 phosphate. Fab1 catalyses the conversion of PtdIns 3 phosphate to PtdIns(3,5)P2 (Michell et al.,2006). In 2003, Gillooly et al. found that PtdIns (3,5)P2 are synthesised in the endosomal system by the action of type III PPIn 3-kinase, suggesting that PtdIns (3,5)P2 plays a role in membrane trafficking in the endosomal and lysosomal areas of the cell.

The main function of PtdIns polyphosphate in the process of membrane trafficking is to transport the protein to a specific regions of the membrane. VPS34p is localised in the cytosol but can be transported to the endosomal membrane by the action of VPS15p (Stack et al., 1993).
When the vps15 gene mutates in such a way that it can no longer function as kinase, the result is a massive decrease in the production of PtdIns 3P and causes defects in vacuolar protein sorting (VPS). The strains that had a deleted vps15 or vps34 gene displayed common phenotypes (Herman et al., 1992). This suggests that the gene product of these genes may follow the same steps as in vacuolar protein sorting (Stack et al., 1993). Also, overproduction of VPS34p in a strain containing a mutated vps15 gene suppressed the growth and protein sorting of vacuolar protein (Herman et al., 1991).

As previously described, PtdIns are converted to PtdIns 3 phosphate by the action of type III PPIn 3-kinase, VPS34p and then to PtdIns (3,5)P2 by the action of PIPKIII, Fab1p (Michell et al, 2006). The corrective functioning of Fab1 requires three additional proteins which are called the activator proteins of Fab1, Vac14p, Vac7p and Fig4p. The cells lacking these activator proteins show all or the same phenotypic defects as shown by cells lacking the fab1 gene (Dove et al., 2002; Duex et al., 2006).

Fig4p is a PPin phosphatase that dephosphorylates PtdIns(3,5)P2 at the 5th position on the inositol ring and degrades it into PtdIns 3P (Gary et al., 2002). In 2004, Rudge et al. created a recombinant form of Fig4p and found out that Fig4p attacks only PtdIns(3,5)P2 among the other PtdIns. Strains lacking the Fig4 gene had a substantial decrease in the production of PtdIns(3,5)P2, suggesting defective PtdIns(3,5)P2 synthesis. This puzzle was solved in recent studies showing that a large complex regulated the synthesis and degradation of PtdIns(3,5)P2. This is formed by Fab1p, vac14 and Fig4p (Dong, 2010; Botelho et al., 2008; Jin et al., 2008).

The common phenotypes that are associated with the yeast strains lack vac14 or vac7. These are the activators of Fab1p. Strains expressing Fab1p that can no longer function as a kinase, or those lacking the effectors of PtdIns(3,5)P2, have an enlarged vacuole that fills almost all of the cell. This suggests that PtdIns(3,5)P2 is required for the membrane and membrane-bound protein recycling (Michell et al., 2005). An enlarged vacuole is also observed in the vpsΔ cells. It is also observed in Schizosaccharomyces pombe when the ste12 gene has been deleted, which acts as a PIPkIII (Onishi et al., 2003; Takegawa et al., 2003). It was observed in Schizosaccharomyces pombe that the mating response to the mating factors slowed down (Michell et al., 2006). The cells were inefficient in mating, meiosis or sporulation due to the slowing down of the endocytic and exocytic membrane trafficking (Michell et al., 2006). These strains also showed inappropriate sensitivity to stresses like heat. This can be overcome by supplementing the growth medium with an abundance of osmolyte. Osmolyte is non-nutritional and corrects the defects in sporulation (Michell et al., 2006). A possible explanation for these effects is that they are due to defective cell-wall assembly that is caused by faulty membrane trafficking (Michell et al., 2006).
PPIns mediate their role on cellular activities by acting as membrane binding sites for various distinct effector proteins. PROPPIN, a family of effector proteins of PtdIns(3,5)P2, comprises tg18p/Svp1p, Atg21p/ Hsv 1p and Hsv 2p in yeast, which controls membrane trafficking between late endosome, MVB and vacuoles (Michell et al., 2006). In 2004, Dove et al. found out that Atg18p/ Svp1p has a high specificity for binding to PtdIns(3,5)P2 and plays a role in membrane trafficking from the vacuole. It was observed in Atg18pΔ cells that there was an excess of PtdIns(3,5)P2 even when it was not under stress (Michell et al., 2006). The strains containing a mutated form of Atg18p, which could not bind to PtdIns(3,5)P2, were found to have defective membrane trafficking from vacuole to endosome and vice versa (Dove et al., 2004).
In 2003, Fraint et al. found an epsin-like protein, Ent3, that plays a role as an effector protein which has a binding specificity to PtdIns(3,5)P2. The role of Ent3 has been observed in the membrane trafficking of cargo proteins between the trans-Golgi network and the vacuole (Hicke et al., 2003). Under normal conditions or a stressed state, the fusion or fragmentation of a vacuole or lysosome is one of the major functions performed by PtdIns(3,5)P2. Conformational changes in the vacuole can be studied by inducing hypo- or hyper-osmotic shock. These studies will help us to get a clear understanding of the role of PtdIns(3,5)P2 in cellular activities. The cellular levels of PtdIns(3,5)P2 increases with hyper-osmotic shock in Saccharomyces cerevisiae. This has also been observed in other organisms, including plants (Dove et al., 1997; Rugde et al., 2004). While moderate increases in the molecular levels of PtdIns(3,5)P2 was observed under modest stress and little vacuole fragmentation was observed, this could be reversed within ten minutes. However, when extensive stress was induced, a dramatic increase in the levels of PtdIns(3,5)P2 was observed with extensive fragmentation of vacuole. This took 60 minutes to reverse (Duex et al., 2006; Dove et al., 2009; Dong.,2010).

There are three fab1 mutants which help to develop a corresponding relationship between vacuole fragmentation and synthesis of PtdIns(3,5)P2:a fab1 mutant that can no longer function as a kinase (D2134R), had an enlarged vacuole (Gary et al., 1998; Odorizzi et al., 1998); FAB1-5 that produces an extraordinarily large amount of PtdIns(3,5)P2 and had a fragmented or shrunken vacuole; and a fab1 mutant which showed little fragmentation of the vacuole (Gary et al., 1998; Odorizzi et al., 1998) as it maintains only 10% of PtdIns(3,5)P2 levels (Dong.,2010).

1.2 Arrestin: Adaptors for Ubiquitin membrane trafficking

Changes in the cell membrane are brought about by the eukaryotic cells for the correct sensing of- and response to environmental cues. Yeast plasma membranes are remodelled either by degrading or recycling the plasma-membrane transporters by the process of endocytosis. Endocytosis is a mechanism by which the cells change their plasma membrane proteins which help in cell growth and differentiation. Ubiquitination is required for endocytosis which leads to vacuole degradation. In almost of the cases reported to date, Rsp5, a ubiquitin ligase is deployed near the plasma membrane (Hicki et al., 2003).
There are 3 WW domains present on Rsp5 that recognise the PY motif with a specific sequence: PPXY or LPXY (Nikko et al., 2009). Various adaptors have been discovered that take part in ubiquitination of protein including Bsd2 (Hettma et al., 2004), Tre1/2 (Stimpson et al., 2006) and soluble proteins Bul1 and Bul2 (Helliwell., 2001; Soetens et al., 2001). Recent work has found that members of yeast arrestin-like proteins take part in the endocytosis of the metal transporter Smt1 and various amino acid transporters including Can1, Mup1 and Lyp1 under stressed condition or in presence of respective amino acids (Lin et al., 2008; Nikko et al.,2008).


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