Although phosphatidylinositol-4-phosphate (PtdIns4P) is known to be an essential regulator of constitutive secretion from the late Golgi in yeast, its role in mammalian cells has remained unclear. It was thought to just be a substrate for the synthesis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), a known essential regulator of plasma-membrane trafficking. However, the Golgi contains only low levels of PtdIns(4,5)P2 and no direct role for PtdIns(4,5)P2 in Golgi-membrane trafficking has been established. So, could PtdIns4P itself have a more important role in the Golgi? In Cell, Yin and colleagues now show that the answer to this question is yes.

The authors first established that the phosphatidylinositol 4-kinase (PI4K) PI4KIIα — which makes PtdIns4P — is a Golgi resident enzyme, and then used small interfering RNA (siRNA) to study the role of this enzyme in the mammalian Golgi. They showed that PI4KIIα siRNA significantly reduced the incorporation of 32P into both PtdIns4P and PtdIns(4,5)P2. Furthermore, they used immunofluorescence to show that PtdIns4P is most concentrated in the Golgi (surprisingly little PtdIns4P was present at the plasma membrane), and that PI4KIIα siRNA selectively decreased these Golgi PtdIns4P levels.

Next, Yin and co-workers showed that PI4KIIα siRNA markedly reduced the association of γ-adaptin — a subunit of the adaptor protein (AP)-1 complex — with the Golgi. AP-1 regulates clathrin-coated-vesicle trafficking between the trans-Golgi network and the endosome/lysosome system, so, as expected, the authors found that the levels of Golgi clathrin were also significantly reduced. By contrast, β-COP — a subunit of the coatomer protein (COP)I complex — remained associated with the Golgi.

The decreased association of γ-adaptin with the Golgi after the addition of PI4KIIα siRNA could be due to a lack of PtdIns4P or PtdIns(4,5)P2, so the authors used a solid-phase, lipid-binding assay to determine which lipid regulates the recruitment of AP-1 to the Golgi. They found that AP-1 binds PtdIns4P, but not PtdIns(4,5)P2, so PtdIns4P is probably the main recruiter of AP-1. Interestingly, this means that the lipid specificities of AP-1 and AP-2 are different — AP-2 preferentially binds PtdIns(4,5)P2 in the plasma membrane.

To verify that PtdIns4P is the main recruiter of AP-1 to the Golgi in vivo, the authors used three methods. First, they shuttled PtdIns4P or PtdIns(4,5)P2 into live cells using membrane-permeant polyamine carriers after the addition of PI4KIIα siRNA. They found that shuttling PtdIns4P into cells rescued AP-1 recruitment, whereas shuttling PtdIns(4,5)P2 did not. They saw the same effect using their second method, in which exogenous PtdIns4P or PtdIns(4,5)P2 was added back to semi-intact cells after PI4KIIα siRNA. In the third method, they showed that anti-PtdIns4P antibodies selectively inhibited AP-1 Golgi recruitment. These data show that PtdIns4P is a crucial Golgi lipid that functions independently of PtdIns(4,5)P2.

On the basis of this work, Yin and colleagues propose that “...PI4KIIα establishes the Golgi's unique lipid-defined organelle identity by generating [PtdIns4P]-rich domains that specify the docking of the AP-1 coat machinery”. In addition, they propose that, as the plasma membrane is enriched in PtdIns(4,5)P2 and endosomes are enriched in phosphatidylinositol-3-phosphate, Golgi PtdIns4P might represent “...the third arm of the membrane lipid recognition system”.