Joseph P. Albanesi

Phosphatidylinositol 4 Phosphate Regulates Targeting of Clathrin Adaptor AP-1 Complexes to the Golgi

Phosphatidylinositol 4 phosphate [PI(4)P] is essential for secretion in yeast, but its role in mammalian cells is unclear. Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator. We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions. This AP-1 binding defect is rescued by adding back PI(4)P. In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that PI4KIIalpha establishes the Golgis unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.

Membrane Fusion: Stalk Model Revisited

Membrane fusion is believed to proceed via intermediate structures called stalks. Mathematical analysis of the stalk provided the elastic energy involved in this structure and predicted the possible evolution of the overall process, but the energies predicted by the original model were suspiciously high. This was due to an erroneous assumption, i.e., that the stalk has a figure of revolution of a circular arc. Here we abandon this assumption and calculate the correct shape of the stalk. We find that it can be made completely stress free and, hence, its energy, instead of being positive and high can become negative, thus facilitating the fusion process. Based on our new calculations, the energies of hemifusion, of complete fusion, and of the pore in a bilayer were analyzed. Implications for membrane fusion and lipid phase transitions are discussed.

Essential role of the dynamin pleckstrin homology domain in receptor-mediated endocytosis.

ABSTRACT Pleckstrin homology (PH) domains are found in numerous membrane-associated proteins and have been implicated in the mediation of protein-protein and protein-phospholipid interactions. Dynamin, a GTPase required for clathrin-dependent endocytosis, contains a PH domain which binds to phosphoinositides and participates in the interaction between dynamin and the βγ subunits of heterotrimeric G proteins. The PH domain is essential for expression of phosphoinositide-stimulated GTPase activity of dynamin in vitro, but its involvement in the endocytic process is unknown. We expressed a series of dynamin PH domain mutants in cultured cells and determined their effect on transferrin uptake by those cells. Endocytosis is blocked in cells expressing a PH domain deletion mutant and a point mutant that fails to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In contrast, expression of a point mutant with unimpaired PI(4,5)P2 interaction has no effect on transferrin uptake. These results demonstrate the significance of the PH domain for dynamin function and suggest that its role may be to mediate interactions between dynamin and phosphoinositides.

A novel family of phosphatidylinositol 4-kinases conserved from yeast to humans.

Phosphatidylinositolpolyphosphates (PIPs) are centrally involved in many biological processes, ranging from cell growth and organization of the actin cytoskeleton to endo- and exocytosis. Phosphorylation of phosphatidylinositol at the D-4 position, an essential step in the biosynthesis of PIPs, appears to be catalyzed by two biochemically distinct enzymes. However, only one of these two enzymes has been molecularly characterized. We now describe a novel class of phosphatidylinositol 4-kinases that probably corresponds to the missing element in phosphatidylinositol metabolism. These kinases are highly conserved evolutionarily, but unrelated to previously characterized phosphatidylinositol kinases, and thus represent the founding members of a new family. The novel phosphatidylinositol 4-kinases, which are widely expressed in cells, only phosphorylate phosphatidylinositol, are potently inhibited by adenosine, but are insensitive to wortmannin or phenylarsine oxide. Although they lack an obvious transmembrane domain, they are strongly attached to membranes by palmitoylation. Our data suggest that independent pathways for phosphatidylinositol 4-phosphate synthesis emerged during evolution, possibly to allow tight temporal and spatial control over the production of this key signaling molecule.

Mechanism of caveolin filament assembly

Caveolin-1 was the first protein identified that colocalizes with the ≈10-nm filaments found on the inside surface of caveolae membranes. We have used a combination of electron microscopy (EM), circular dichroism, and analytical ultracentrifugation to determine the structure of the oligomers that form when the first 101 aa of caveolin-1 (Cav1–101) are allowed to associate. We determined that amino acids 79–96 in this caveolin-1 fragment are arranged in an α-helix. Cav1–101 oligomers are ≈11 nm in diameter and contain seven molecules of Cav1–101. These subunits, in turn, are able to assemble into 50 nm long × 11 nm diameter filaments that closely match the morphology of the filaments in the caveolae filamentous coat. We propose that the heptameric subunit forms in part through lateral interactions between the α-helices of the seven Cav1–101 units. Caveolin-1, therefore, appears to be the structural molecule of the caveolae filamentous coat.

Membrane guanylyl cyclase receptors: an update

Recent studies have demonstrated key roles for several membrane guanylyl cyclase receptors in the regulation of cell hyperplasia, hypertrophy, migration and extracellular matrix production, all of which having an impact on clinically relevant diseases, including tissue remodeling after injury. Additionally, cell differentiation, and even tumor progression, can be profoundly influenced by one or more of these receptors. Some of these receptors also mediate important communication between the heart and intestine, and the kidney to regulate blood volume and Na+ balance.

Phosphatidylinositol (4,5)-Bisphosphate-dependent Activation of Dynamins I and II Lacking the Proline/Arginine-rich Domains

Dynamins comprise a family of GTPases that participate in the early stages of endocytosis. The GTPase activity of neuronal specific dynamin I is stimulated by microtubules, negatively charged phospholipid vesicles, and Src homology 3-containing proteins, including Grb2. These activators were previously shown to bind to a proline/arginine-rich domain (PRD) in the carboxyl-terminal region of the enzyme. Dynamin II, which is ubiquitously expressed, had not been purified or characterized previously. In this study, the enzymatic properties of rat dynamin II and of D746, a dynamin II truncation mutant lacking the PRD, have been characterized. Dynamin II has a higher basal activity than dynamin I, but the two types of dynamin are stimulated similarly by microtubules, Grb2, and phospholipids. D746 is not activated by microtubules or Grb2, highlighting the significance of the PRD for these interactions, but it is activated by phospholipid vesicles containing phosphatidylserine or phosphatidylinositol-4,5- bisphosphate. Moreover, in contrast to previous reports, the PRD appears not to be required for phospholipid-stimulated self-assembly of dynamin, which is a key element in the regulation of its activity. Similar results were obtained with bovine brain dynamin I that had been subjected to limited proteolytic digestion to remove the PRD. Our data highlight the potential involvement of dynamin pleckstrin homology domains in the regulation of GTPase activity by phospholipids.

Palmitoylation Controls the Catalytic Activity and Subcellular Distribution of Phosphatidylinositol 4-Kinase IIα

Phosphatidylinositol 4-kinases play essential roles in cell signaling and membrane trafficking. They are divided into type II and III families, which have distinct structural and enzymatic properties and are essentially unrelated in sequence. Mammalian cells express two type II isoforms, phosphatidylinositol 4-kinase IIα (PI4KIIα) and IIβ (PI4KIIβ). Nearly all of PI4KIIα, and about half of PI4KIIβ, associates integrally with membranes, requiring detergent for solubilization. This tight membrane association is because of palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domains of both type II isoforms. Deletion of this motif from PI4KIIα converts the kinase from an integral to a tightly bound peripheral membrane protein and abrogates its catalytic activity ( Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708 ). Here we identify the first two cysteines in the CCPCC motif as the principal sites of palmitoylation under basal conditions, and we demonstrate the importance of the central proline for enzymatic activity, although not for membrane binding. We further show that palmitoylation is critical for targeting PI4KIIα to the trans-Golgi network and for enhancement of its association with low buoyant density membrane fractions, commonly termed lipid rafts. Replacement of the four cysteines in CCPCC with a hydrophobic residue, phenylalanine, substantially restores catalytic activity of PI4KIIα in vitro and in cells without restoring integral membrane binding. Although this FFPFF mutant displays a perinuclear distribution, it does not strongly co-localize with wild-type PI4KIIα and associates more weakly with lipid rafts.

The CD3 ζ Subunit Contains a Phosphoinositide-Binding Motif That Is Required for the Stable Accumulation of TCR–CD3 Complex at the Immunological Synapse

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P2, and PtdIns(3,4,5)P3 with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell–APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS–phosphoinositide interactions in supporting T cell activation.

The Cytoplasmic Tail of the T Cell Receptor CD3 ε Subunit Contains a Phospholipid-Binding Motif that Regulates T Cell Functions

The CD3 ε subunit of the TCR complex contains two defined signaling domains, a proline-rich sequence and an ITAM. We identified a third signaling sequence in CD3 ε, termed the basic-rich stretch (BRS). Herein, we show that the positively charged residues of the BRS enable this region of CD3 ε to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and PI(4,5)P2. Transgenic mice containing mutations of the BRS exhibited varying developmental defects, ranging from reduced thymic cellularity to a complete block in T cell development. Peripheral T cells from BRS-modified mice also exhibited several defects, including decreased TCR surface expression, reduced TCR-mediated signaling responses to agonist peptide-loaded APCs, and delayed CD3 ε localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 ε lipid-binding domain in T cell biology.