Thomas F. J. Martin

PI(4,5)P2 regulation of surface membrane traffic

Phosphatidylinositol 4,5-biphosphate (PI[4,5]P(2)) has emerged as an important signaling molecule in the membrane for regulating vesicle exo- and endocytosis and the accompanying actin cytoskeletal rearrangements. Localization studies with GFP-tagged binding domains and antibodies provide new views of the non-uniform, dynamic distribution of PI(4,5)P(2) in membranes and its organization in raft-like domains. The targeting of phosphoinositide kinases by GTPases can coordinate the reactions of membrane fusion and fission with cytoskeletal assembly, providing a basis for membrane movement.

The b alleles of U. maydis, whose combinations program pathogenic development, code for polypeptides containing a homeodomain-related motif.

U. maydis is a fungal pathogen of corn with two forms: one is yeast-like and nonpathogenic; the other is filamentous and pathogenic. The b locus, with 25 different alleles, regulates this dimorphism: any combination of two different alleles triggers pathogenic development, whereas the presence of identical alleles results in the yeast-like form. We have cloned four b alleles (b1, b2, b3, and b4) and show that the b locus contains a single open reading frame (ORF) of 410 amino acids with a variable N-terminal region and a highly conserved C-terminal region (60% and 93% identity, respectively). Mutational analysis confirms that this ORF is responsible for b activity. The b polypeptides appear to be DNA binding proteins because they contain a motif related to the homeodomain in their constant region. We propose that combinatorial interactions between b polypeptides generate regulatory proteins that determine the developmental program of the fungus.

A novel 145 kd brain cytosolic protein reconstitutes Ca2+-regulated secretion in permeable neuroendocrine cells

The regulated secretory pathway is activated by elevated cytoplasmic Ca2+; however, the components mediating Ca2+ regulation have not been identified. In semi-intact neuroendocrine cells, Ca(2+)-activated secretion is ATP- and cytosol protein-dependent. We have identified a novel brain protein, p145, as a cytosolic factor that reconstitutes Ca(2+)-activated secretion in two neuroendocrine cell types. The protein is a dimer of 145 kd subunits, exhibits Ca(2+)-dependent interaction with a hydrophobic matrix, and binds phospholipid vesicles, suggesting a membrane-associated function. A p145-specific antibody inhibits the reconstitution of Ca(2+)-activated secretion by cytosol, indicating an essential role for p145. The restricted expression of p145 in tissues exhibiting a regulated secretory pathway suggests a key role for this protein in the transduction of Ca2+ signals into vectorial membrane fusion events.

Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis.

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.

Different domains of synaptotagmin control the choice between kiss-and-run and full fusion

Exocytosis—the release of the contents of a vesicle—proceeds by two mechanisms. Full fusion occurs when the vesicle and plasma membranes merge. Alternatively, in what is termed kiss-and-run, vesicles can release transmitter during transient contacts with the plasma membrane. Little is known at the molecular level about how the choice between these two pathways is regulated. Here we report amperometric recordings of catecholamine efflux through individual fusion pores. Transfection with synaptotagmin (Syt) IV increased the frequency and duration of kiss-and-run events, but left their amplitude unchanged. Endogenous Syt IV, induced by forskolin treatment, had a similar effect. Full fusion was inhibited by mutation of a Ca2+ ligand in the C2A domain of Syt I; kiss-and-run was inhibited by mutation of a homologous Ca2+ ligand in the C2B domain of Syt IV. The Ca2+ sensitivity for full fusion was 5-fold higher with Syt I than Syt IV, but for kiss-and-run the Ca2+ sensitivities differed by a factor of only two. Syt thus regulates the choice between full fusion and kiss-and-run, with Ca2+ binding to the C2A and C2B domains playing an important role in this choice.

PIP Kinase Iγ Is the Major PI(4,5)P2 Synthesizing Enzyme at the Synapse

Abstract Disruption of the presynaptically enriched polyphosphoinositide phosphatase synaptojanin 1 leads to an increase of clathrin-coated intermediates and of polymerized actin at endocytic zones of nerve terminals. These changes correlate with elevated levels of PI(4,5)P 2 in neurons. We report that phosphatidylinositol phosphate kinase type Iγ (PIPKIγ), a major brain PI(4)P 5-kinase, is concentrated at synapses. Synaptojanin 1 and PIPKIγ antagonize each other in the recruitment of clathrin coats to lipid membranes. Like synaptojanin 1 and other proteins involved in endocytosis, PIPKIγ undergoes stimulation-dependent dephosphorylation. These results implicate PIPKIγ in the synthesis of a PI(4,5)P 2 pool that acts as a positive regulator of clathrin coat recruitment and actin function at the synapse.

ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIγ restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIγ with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.

N-ETHYLMALEIMIDE-SENSITIVE FACTOR ACTS AT A PREFUSION ATP-DEPENDENT STEP IN CA2+-ACTIVATED EXOCYTOSIS

An ATP-dependent activity of NSF (N-ethylmaleimide-sensitive factor) that rearranges soluble NSF attachment protein (SNAP) receptor (SNARE) protein complexes was proposed to be the driving force for membrane fusion. The Ca2+-activated fusion of secretory vesicles with the plasma membrane in permeable PC12 cells requires ATP; however, the ATP requirement is for a priming step that precedes the Ca2+-triggered fusion reaction. While phosphoinositide phosphorylation is a key reaction required for priming, additional ATP-dependent reactions are also necessary. Here we report that the NSF-catalyzed rearrangement of SNARE protein complexes occurs during ATP-dependent priming. NSF with α-SNAP (soluble NSF attachment protein) were required for ATP-dependent priming but not Ca2+-triggered fusion, indicating that NSF acts at an ATP-dependent prefusion step rather than at fusion itself. NSF-catalyzed activation of SNARE proteins may reorganize membranes to generate a vesicle-plasma membrane prefusion intermediate that is poised for conversion to full fusion by Ca2+-dependent mechanisms.

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P2) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P2 as ∼6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P2 soluble NSF attachment protein receptor (SNARE)–dependent liposome fusion is inhibited. Inhibition by PI 4,5-P2 likely results from its intrinsic positive curvature–promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P2, suggesting that syntaxin sequesters PI 4,5-P2 to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P2, we test a PI 4,5-P2–binding priming factor required for vesicle exocytosis. Ca2+-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P2 dependent. These results indicate that PI 4,5-P2 regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

SNAP-25 Is Required for a Late Postdocking Step in Ca2+-dependent Exocytosis

The Ca2+-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca2+-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile181-Gln197) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca2+-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.