James G. Alb

Yeast Sec14p Deficient in Phosphatidylinositol Transfer Activity Is Functional In Vivo

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity. As expected, Sec14pK66,239A fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo. However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1ts and sec14 null mutations. This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs. These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.

Functional Characterization of a Mammalian Sac1 and Mutants Exhibiting Substrate-specific Defects in Phosphoinositide Phosphatase Activity

The Saccharomyces cerevisiae SAC1gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex. Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral membrane phosphoinositide phosphatases, the synaptojanins, and these Sac1 domains define novel phosphoinositide phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic phosphoinositide phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in Δsac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3,5-bisphosphate phosphatase activities, is essential for the heterologous complementation of Sac1p defectsin vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.

Phospholipid transfer activity is relevant to but not sufficient for the essential function of the yeast SEC14 gene product.

To investigate several key aspects of phosphatidylinositol transfer protein (PI‐TP) function in eukaryotic cells, rat PI‐TP was expressed in yeast strains carrying lesions in SEC14, the structural gene for yeast PI‐TP (SEC14p), whose activity is essential for Golgi secretory function in vivo. Rat PI‐TP expression effected a specific complementation of sec14ts growth and secretory defects. Complementation of sec14 mutations was not absolute as rat PI‐TP expression failed to rescue sec14 null mutations. This partial complementation of sec14 lesions by rat PI‐TP correlated with inability of the mammalian protein to stably associate with yeast Golgi membranes and was not a result of rat PI‐TP stabilizing the endogenous sec14ts gene product. These collective data demonstrate that while the in vitro PI‐TP activity of SEC14p clearly reflects some functional in vivo property of SEC14p, the PI‐TP activity is not the sole essential activity of SEC14p. Those data further identify an efficient Golgi targeting capability as a likely essential feature of SEC14p function in vivo. Finally, the data suggest that stable association of SEC14p with yeast Golgi membranes is not a simple function of its lipid‐binding properties, indicate that the amino‐terminal 129 SEC14p residues are sufficient to direct a catalytically inactive form of rat PI‐TP to the Golgi and provide the first evidence to indicate that a mammalian PI‐TP can stimulate Golgi secretory function in vivo.

Mice Lacking Phosphatidylinositol Transfer Protein-α Exhibit Spinocerebellar Degeneration, Intestinal and Hepatic Steatosis, and Hypoglycemia

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and cellular functions. We now report that ablation of PITPα function leads to aponecrotic spinocerebellar disease, hypoglycemia, and intestinal and hepatic steatosis in mice. The data indicate that hypoglycemia is in part associated with reduced proglucagon gene expression and glycogenolysis that result from pancreatic islet cell defects. The intestinal and hepatic steatosis results from the intracellular accumulation of neutral lipid and free fatty acid mass in these organs and suggests defective trafficking of triglycerides and diacylglycerols from the endoplasmic reticulum. We propose that deranged intestinal and hepatic lipid metabolism and defective proglucagon gene expression contribute to hypoglycemia in PITPα–/– mice, and that hypoglycemia is a significant contributing factor in the onset of spinocerebellar disease. Taken together, the data suggest an unanticipated role for PITPα in with glucose homeostasis and in mammalian endoplasmic reticulum functions that interface with transport of specific luminal lipid cargoes.

A Phosphatidylinositol 3-Kinase and Phosphatidylinositol Transfer Protein Act Synergistically in Formation of Constitutive Transport Vesicles from the Trans-Golgi Network

Current evidence suggests that phosphatidylinositol (PI) kinases and phosphatidylinositol transfer protein (PITP) are involved in driving vesicular traffic from yeast and mammalian trans-Golgi network (TGN). We have tested the interaction between these cytosolic proteins in an assay that measures the formation of constitutive transport vesicles from the TGN in a hepatocyte cell-free system. This reaction is dependent on a novel PI 3-kinase, and we now report that, under conditions of limiting cytosol, purified PI 3-kinase and PITP functionally cooperate to drive exocytic vesicle formation. This synergy was observed with both yeast and mammalian PITPs, and it also extended to the formation of PI 3-phosphate. These collective findings indicate that the PI 3-kinase and PITP synergize to form a pool of PI 3-phosphate that is essential for formation of exocytic vesicles from the hepatocyte TGN.

Phosphatidylinositol transfer proteins: the long and winding road to physiological function

Phosphatidylinositol transfer proteins (PITPs) have historically been thought to help execute lipid-sorting events by transporting phospholipid monomers between membrane bilayers. Recent data, however, indicate unanticipated roles for PITPs in the coordination and/or coupling of phospholipid metabolism with vesicle trafficking and the downregulation of signal-transduction reactions. We are only now beginning to appreciate both the identities of PITP-dependent cellular reactions and the intriguing mechanisms by which PITPs execute their functions in eukaryotic cells.

Phospholipid metabolism and membrane dynamics

Genetic and biochemical approaches are shedding new light on the distinct physiological functions of specific phospholipid metabolic pathways and the mechanisms by which phospholipids are mobilized between intracellular compartments. In particular, phosphatidylinositol-transfer proteins have recently been revealed to play fascinating and unanticipated roles in the coordination of phospholipid metabolism with vesicle-trafficking and signal-transducing reactions.

Zebrafish Class 1 Phosphatidylinositol Transfer Proteins: PITPβ and Double Cone Cell Outer Segment Integrity in Retina

Phosphatidylinositol transfer proteins (PITPs) in yeast co‐ordinate lipid metabolism with the activities of specific membrane trafficking pathways. The structurally unrelated metazoan PITPs (mPITPs), on the other hand, are an under‐investigated class of proteins. It remains unclear what biological activities mPITPs discharge, and the mechanisms by which these proteins function are also not understood. The soluble class 1 mPITPs include the PITPα and PITPβ isoforms. Of these, the β‐isoforms are particularly poorly characterized. Herein, we report the use of zebrafish as a model vertebrate for the study of class 1 mPITP biological function. Zebrafish express PITPα and PITPβ‐isoforms (Pitpna and Pitpnb, respectively) and a novel PITPβ‐like isoform (Pitpng). Pitpnb expression is particularly robust in double cone cells of the zebrafish retina. Morpholino‐mediated protein knockdown experiments demonstrate Pitpnb activity is primarily required for biogenesis/maintenance of the double cone photoreceptor cell outer segments in the developing retina. By contrast, Pitpna activity is essential for successful navigation of early developmental programs. This study reports the initial description of the zebrafish class 1 mPITP family, and the first analysis of PITPβ function in a vertebrate.

The pathologies associated with functional titration of phosphatidylinositol transfer protein α activity in mice

Phosphatidylinositol transfer proteins (PITPs) bind phosphatidylinositol (PtdIns) and phosphatidylcholine and play diverse roles in coordinating lipid metabolism/signaling with intracellular functions. The underlying mechanisms remain unclear. Genetic ablation of PITPα in mice results in neonatal lethality characterized by intestinal and hepatic steatosis, spinocerebellar neurodegeneration, and glucose homeostatic defects. We report that mice expressing a PITPα selectively ablated for PtdIns binding activity (PitpαT59D), as the sole source of PITPα, exhibit phenotypes that recapitulate those of authentic PITPα nullizygotes. Analyses of mice with graded reductions in PITPα activity reveal proportionately graded reductions in lifespan, demonstrate that intestinal steatosis and hypoglycemia are apparent only when PITPα protein levels are strongly reduced (⩾90%), and correlate steatotic and glucose homeostatic defects with cerebellar inflammatory disease. Finally, reconstitution of PITPα expression in the small intestine substantially corrects the chylomicron retention disease and cerebellar inflammation of Pitpα0/0 neonates, but does not rescue neonatal lethality in these animals. These data demonstrate that PtdIns binding is an essential functional property of PITPα in vivo, and suggest a causal linkage between defects in lipid transport and glucose homeostasis and cerebellar inflammatory disease. Finally, the data also demonstrate intrinsic neuronal deficits in PITPα-deficient mice that are independent of intestinal lipid transport defects and hypoglycemia.

Lamellipodia are crucial for haptotactic sensing and response

ABSTRACT Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis – differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments. Highlighted Article: Haptotaxis (directed migration) on a substrate-bound gradient is perhaps the least-well understood form of directed migration. We show that differential lamellipodial dynamics are crucial for this process.