Jcb_201404041 1..7

Phosphoinositide signaling modules play fundamental roles in intracellular signaling pathways and in defining organelle identity. They are composed of a phosphatidylinositol kinase, effector proteins that specifically recognize its product, and a phosphatase that terminates signaling. The phosphatidylinositol 4-phosphate (PtdIns4P) signaling modules that function at the Golgi apparatus control the lipid content of Golgi membranes and regulate export of cargo from the trans-Golgi network (TGN; Graham and Burd, 2011; Santiago-Tirado and Bretscher, 2011). PtdIns4P is largely restricted to the TGN and, hence, this lipid is a defining feature of this compartment. In budding yeast, the Golgi phosphatidylinositol 4-kinase (PI4K), called Pik1, localizes to late Golgi compartments and PtdIns4P restriction to the TGN is enforced by a broad specificity phosphatidylinositol phosphatase called Sac1, which localizes to the ER and early Golgi compartments. Sac1 substrates localize to a variety of organelles, including the plasma membrane, endosome, and Golgi apparatus, but the mechanisms by which Sac1 accesses these substrates are poorly understood. An important structural feature of Sac1 is that the Sac1 homology domain (Sac1-HD), which contains the catalytic subdomain, is linked to the membrane-spanning segments via an 70-residue-long sequence that is disordered in the yeast Sac1HD crystal structure that was determined by x-ray crystallography (Manford et al., 2010). It was proposed that the unstructured segment allows the Sac1 catalytic domain to access substrate in cis (i.e., on the same membrane) and also in trans, at regions where the ER is closely apposed to another organelle membrane (Manford et al., 2010; Stefan et al., 2011), though recent work has challenged the view that Sac1 can act in trans (Mesmin et al., 2013). The Sac1-HD is a composite of a catalytic and an intimately associated N-terminal subdomain (Manford et al., 2010). The function of the N-terminal subdomain is still unknown but is likely physiologically important as mutations in this domain in the Sac1-related protein Sac3/Fig4 are proposed to result in several neuropathies (Chow et al., 2007, 2009). We recently discovered that a Golgi-localized PI4K effector called Vps74 binds Sac1-HD (Wood et al., 2012). Vps74 is proposed to mediate packaging of medial Golgi glycosyltransferases into coatomer (also called COP1)-coated vesicles that bud Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1– Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination. Sac1–Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus