The Sac1 Phosphoinositide Phosphatase Regulates Golgi Membrane Morphology and Mitotic Spindle Organization in Mammals

Abstract

Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties. INTRODUCTIONSpatial and temporal regulation of intracellular signaling in eukaryotic cells involves the compartmentalization of membrane surfaces into discrete, albeit often transient, functional units. There are several biochemical strategies by which cells generate such units or domains. One well-established strategy uses the chemical diversity offered by phosphoinositides (PIPs), i.e., phosphorylated forms of phosphatidylinositol (PtdIns) (Majerus, 1997;Fruman et al., 1998;Strahl and Thorner, 2007). The chemical heterogeneity of individual PIP species permits the construction of chemically unique platforms on membrane surfaces that, in turn, recruit unique cohorts of proteins that drive specific biological reactions. Spatial and temporal control of such reactions requires a finely coordinated balance between the activities of the lipid kinases that produce PIPs and the activities of enzymes that degrade them. PIP turnover is catalyzed by two general classes of enzymes: phospholipases and PIP phosphatases.Although experimental scrutiny of the phospholipases has historically been more intense, recent demonstrations of the significant biological functions played by PTEN, the myotubularins, and synaptojanins highlight the general importance of PIP phosphatases (Wishart et al., 2001;Wishart and Dixon, 2002;Wenk and De Camilli, 2004).The SAC domain derives from the yeast Sac1 protein (ySac1; Cleves et al., 1989), and it represents a signature for PIP phosphatase catalytic activity . PIP phosphatases such as phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN) (Maehama et al., 2001), synaptojanins (Cremona et al., 1999), and synaptojanin-like proteins (Srinivasan et al., 1997;Stolz et al., 1998) all harbor SAC domains. The prototypical member of this family, ySac1, catalyzes the dephosphoryla...

Changes in leukocyte gene expression profiles induced by antineoplastic chemotherapy

“…The SACM1L gene encodes a phosphoinositide phosphatase that is an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus representing a class of phosphoinositide degradation enzymes (31,32). The integral membrane Sac1 phosphatases are a major class of this type of enzyme.…”

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…Importantly, using PtdIns4P-recognizing PH domains, no sign of PtdIns4P accumulation in any cellular compartment could be detected, although an increased level of [ 3 H]PtdIns4P was demonstrable in metabolically labeled cells (929). Sac1 knockdown also caused a high level of spindle defects, creating aberrant chromosomal segregation and a block in exit from mitosis (929). All of these defects can be rescued by overexpression of Sac1, and both its catalytic activity and ability to cycle between the ER and the Golgi were required for complementation (929).…”

“…Sac1-deficient cells show a major disorganization of their Golgi morphology without a gross defect in secretion. Importantly, using PtdIns4P-recognizing PH domains, no sign of PtdIns4P accumulation in any cellular compartment could be detected, although an increased level of [ 3 H]PtdIns4P was demonstrable in metabolically labeled cells (929). Sac1 knockdown also caused a high level of spindle defects, creating aberrant chromosomal segregation and a block in exit from mitosis (929).…”

“…This process is reversed by growth factor stimulation via activation of p38 MAPK (147). Mammalian Sac1 is an essential protein, and deletion of the gene in mouse causes preimplantation lethality, and even in cells, knockdown of Sac1 leads to cell death, making it difficult to study the functions of the protein (929). Sac1-deficient cells show a major disorganization of their Golgi morphology without a gross defect in secretion.…”

Phosphoinositide phosphatases and disease

“…Sac1 is a transmembrane protein enriched in endoplasmic reticulum (ER)-Golgi fractions and is involved in ATP transport into the ER, growth factor signaling, and cell shape (13). Loss of sac1 in yeast genetically links to overcoming defects in both actin and secretory mutants (16,17) and in mice results in early embryonic lethality (18). Mutations in FIG4 in mice cause the pale tremor mouse syndrome, while mutation of the human ortholog causes a form of Charcot Marie Tooth disease (19,20) (Table 1).…”