Interaction of α-taxilin Localized on Intracellular Components with the Microtubule Cytoskeleton

Abstract

ABSTRACT. Intracellular vesicle traffic plays an essential role in the establishment and maintenance of organelle identity and biosynthetic transport. We have identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. Recently, we have found that α-taxilin is over-expressed in malignant tissues including hepatocellular carcinoma and renal cell carcinoma. However, a precise role of α-taxilin in intracellular vesicle traffic and carcinogenesis remains unclear. Then, we first investigated here the intracellular distribution of α-taxilin in Hela cells. Immunofluorescence studies showed that α-taxilin distributes throughout the cytoplasm and exhibits a tubulo-vesicular pattern. Biochemical studies showed that α-taxilin is abundantly localized on intracellular components as a peripheral membrane protein. Moreover, we found that α-taxilin distributes in microtubule-dependent and syntaxin-independent manners, that α-taxilin directly binds to polymerized tubulin in vitro, and that N-ethylmaleimide but not brefeldin A affects the intracellular distribution of α-taxilin. These results indicate that α-taxilin is localized on intracellular components in a syntaxin-independent manner and that the α-taxilin-containing intracellular components are associated with the microtubule cytoskeleton and suggest that α-taxilin functions as a linker protein between the α-taxilin-containing intracellular components and the microtubule cytoskeleton.

α-Taxilin Interacts with Sorting Nexin 4 and Participates in the Recycling Pathway of Transferrin Receptor

“…At present, it is not clear how α-taxilin regulates the proper recycling of TfnR. We previously reported that α-taxilin directly interacts with polymerized tubulin [11] and in this study we found that knockdown of α-taxilin decreases the number and length of SNX4-positive tubular structures. Therefore, it is possible that the function of α-taxilin is to connect SNX4-positive tubular structures to microtubules and support the stability of SNX4-positive tubular structures.…”

“…We previously demonstrated that α-taxilin is a peripheral membrane protein [11]. We then examined whether the membrane association of α-taxilin depends on interaction with SNX4.…”

“…However, significant colocalization between EEA1 or HA-Rab11 and α-taxilin was not observed, suggesting that the majority of α-taxilin is not present on the membrane of early endosomes or recycling endosomes. Furthermore, the majority of α-taxilin did not colocalize with SNX4 in immunofluorescence analysis and a small portion of α-taxilin colocalized with α-tubulin [11], suggesting that the interaction between SNX4-positive tubular structures and α-taxilin, and microtubules and α-taxilin may be transient, or that only a limited region of SNX4-positive tubular structures and microtubules may be associated with α-taxilin. In either case, because α-taxilin is distributed throughout the cytoplasm, it is thought that the colocalization of α-taxilin with SNX4 is difficult to detect in our immunofluorescent analysis.…”

“…Subcellular fractionation was performed as described previously [11]. Briefly, HeLaS3 cells transfected with control or SNX4 siRNA were suspended in buffer A [10 mM Tris/HCl (pH 7.5), 1 mM EDTA and 0.25 M sucrose], and homogenized with 15 strokes in a ball homogenizer (clearance: 0.012 mm).…”

“…It was reported that α-taxilin is overexpressed in hepatocellular carcinoma, renal cell carcinoma and glioblastoma [8]–[10]. Furthermore, we recently reported that α-taxilin binds to polymerized tubulin and partly colocalizes with α-tubulin [11], but the precise function of α-taxilin remains to be elucidated.…”