Abstract
We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup. The endophilins B show the same domain organization as the endophilins A, which contain an N-terminal domain responsible for lipid binding and lysophosphatidic acid acyl transferase activity, a central coiled-coil domain for oligomerization, a less conserved linker region, and a C-terminal Src homology 3 (SH3) domain. The endophilin B1 gene gives rise to at least three splice variants, endophilin B1a, which shows a widespread tissue distribution, and endophilins B1b and B1c, which appear to be brain-specific. Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits lysophosphatidic acid acyl transferase activity, and interacts with dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to endophilins A, endophilin B1 does not bind to synaptojanin 1 and synapsin 1, and overexpression of its SH3 domain does not inhibit transferrin endocytosis. Consistent with this, immunofluorescence analysis of endophilin B1b transfected into fibroblasts shows an intracellular reticular staining, which in part overlaps with that of endogenous dynamin. Upon subcellular fractionation of brain and transfected fibroblasts, endophilin B1 is largely recovered in association with membranes. Together, our results suggest that the action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.
Highlights
We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup
In human endophilin B1, whose cDNA sequence was obtained from data base searches and sequencing of expressed sequence tags (ESTs) clones, the putative translation initiation site is located at nucleotides 151–153 and, in contrast to the murine homolog, is preceded by an in-frame stop codon
Like endophilin A1 [8, 15, 16], endophilin B1 binds lipids, exhibits lysophosphatidic acid (LPA)-AT activity, and tubulates liposomes, properties that have been shown or are likely to reside in its LBM domain (Fig. 5) [15]
Summary
The experiments were performed using the Matchmaker 2 two-hybrid system (Clontech). The full-length PACSIN 1 open reading frame. Was cloned in-frame with the GAL4 DNA-binding domain in the pAS2–1 vector and confirmed by sequencing. The pAS2–1 PACSIN 1 plasmid was sequentially cotransformed with the Matchmaker mouse brain cDNA library into the yeast strain Y190. Transformed yeast cells expressing interacting Gal fusion proteins were selected by the ability to grow on minimal synthetic dropout medium lacking L-tryptophan, L-leucine, and L-histidine and checked for their ability to express the lacZ gene by -galactosidase filter assays. Positive clones were isolated by re-streaking and growth on cycloheximide-containing medium and verified for the interaction with PACSIN 1 by mating with Y187 yeast cells transformed with pAS2–1 PACSIN 1. DNA and protein sequence analyses were performed using the GCG software package (University of Wisconsin, Madison, WI), and multiple gene databases were searched using BLAST programs [23]
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