Interaction of the taxilin family with the nascent polypeptide-associated complex that is involved in the transcriptional and translational processes.

alpha-Taxilin is a novel binding partner of the syntaxin family, which is implicated in intracellular vesicle traffic. We have here found that alpha-taxilin interacts with the nascent polypeptide-associated complex (NAC), which is involved in transferring growing nascent polypeptide chains to appropriate co-translationally acting factors. NAC is composed of two subunits, alpha- and betaNACs. Both these subunits bound to alpha-taxilin through its C-terminal coiled-coil region in dose-dependent and saturable manners. The interactions of alpha-taxilin with alphaNAC and NAC but not with betaNAC were inhibited by syntaxin-4, indicating that alpha-taxilin binds to NAC mainly through its interaction with alphaNAC. When alphaNAC was over-expressed in COS-7 cells, alphaNAC was distributed in the cytosol and nucleus. However, co-expression of the alpha-taxilin fragment containing the alphaNAC-binding region eliminated the nuclear distribution of over-expressed alphaNAC. Moreover, other taxilin family members, beta- and gamma-taxilins, also bound to alphaNAC and thereby affected the nuclear distribution of over-expressed alphaNAC. Taken together with the evidence that alphaNAC functions in the nucleus as a transcriptional coactivator, our results raise the possibility that the taxilin family is involved not only in the translational process through its interaction with NAC but also in the transcriptional process through its interaction with alphaNAC alone.

Identification and characterization of taxilin isoforms.

The syntaxin family is implicated in intracellular vesicle traffic. We have recently identified taxilin, a novel syntaxin-binding protein, which has a long coiled-coil region in its C-terminal half. A database search has revealed the presence of two other molecules having a long coiled-coil region homologous to that of taxilin in mammals. Then, we here attempted to isolate and characterize the two molecules. Both the two molecules stoichiometrically interacted with several syntaxin family members. Then, we renamed original taxilin alpha-taxilin and named the two molecules beta- and gamma-taxilins, respectively. Beta-taxilin was a human homologue of chicken MDP77. Gamma-taxilin was an uncharacterized protein and Northern blot analysis revealed that gamma-taxilin was ubiquitously expressed. Beta- and gamma-taxilins preferentially interacted with syntaxin-1a and -4, respectively. The taxilin family members mutually interacted with the syntaxin family members. These results indicate that there is the taxilin family composed of at least three members in mammals.

Interaction of taxilin with syntaxin which does not form the SNARE complex.

Taxilin is novel binding partner of the syntaxin family, which is implicated in intracellular vesicle traffic. However, precise binding properties of taxilin to the syntaxin family remain to be clarified. Then, we here further investigated the interaction of taxilin with the syntaxin family by use of recombinant taxilins. Syntaxin-1a, -3, and -4 bound to taxilin in a dose-dependent and saturable manners. The concentrations of syntaxin-1a, -3, and -4 giving a half-maximal binding to taxilin were about 1.5, 3.0, and 1.0microM, respectively. The interaction of taxilin with syntaxin-1a was inhibited by SNAP-25 or Munc18 in a dose-dependent manner. When recombinant taxilin was incubated with the extract from the rat brain crude membrane fraction, recombinant taxilin bound to syntaxin-1s free of at least VAMP2, SNAP-25, and Munc18. These results suggest that taxilin interacts with the syntaxin family which does not form at least the SNARE complex.

Taxilin; a novel syntaxin-binding protein that is involved in Ca2+-dependent exocytosis in neuroendocrine cells.

BACKGROUND: The syntaxin family is a central coordinator and participates in multiple protein-protein interactions in the soluble N-ethyl maleimide-sensitive factor attachment protein receptor machinery, which is involved in intracellular vesicle traffic. However, the molecular mechanism by which the syntaxin family regulates intracellular vesicle transport is not well known. RESULTS: We have identified and purified a novel binding partner of syntaxin-3 from rat lung, and isolated and sequenced the cDNA of its human homologue from a human brain cDNA library. The cDNA had an open reading frame encoding a protein of 546 amino acids with a calculated Mr of 61,890. We tentatively referred to this protein as taxilin. A structural analysis of taxilin revealed the existence of an extraordinarily long coiled-coil domain in its C-terminal half. Syntaxin-1a and -4, as well as syntaxin-3 interacted with taxilin, but syntaxin-7 or -8 did not. Northern blot analysis showed that taxilin was ubiquitously expressed. Over-expression of full-length taxilin inhibited Ca2+-dependent exocytosis in PC12 cells. CONCLUSIONS: These results indicate that taxilin is a novel binding partner of several syntaxin family members and suggest that taxilin is involved in Ca2+-dependent exocytosis in neuroendocrine cells.