Abstract
The membrane-bound form of acetylcholinesterase (AChE) constitutes the major component of this enzyme in the mammalian brain. These molecules are hetero-oligomers, composed of four AChE catalytic subunits of type T (AChE(T)), associated with a transmembrane protein of type 1, called PRiMA (proline-rich membrane anchor). PRiMA consists of a signal peptide, an extracellular domain that contains a proline-rich motif (14 prolines with an intervening leucine, P4LP10), a transmembrane domain, and a cytoplasmic domain. Expression of AChE(T) subunits in transfected COS cells with a truncated PRiMA, without its transmembrane and cytoplasmic domains (P(stp54) mutant), produced secreted heteromeric complexes (T4-P(stp54)), instead of membrane-bound tetramers. In this study, we used a series of deletions and point mutations to analyze the interaction between the extracellular domain of PRiMA and AChE(T) subunits. We confirmed the importance of the polyproline stretches and defined a peptidic motif (RP4LP10RL), which induces the assembly and secretion of a heteromeric complex with four AChE(T) subunits, nearly as efficiently as the entire extracellular domain of PRiMA. It is noteworthy that deletion of the N-terminal segment preceding the prolines had little effect. Interestingly, short PRiMA mutants, truncated within the proline-rich motif, reduced both cellular and secreted AChE activity, suggesting that their interaction with AChE(T) subunits induces their intracellular degradation.
Highlights
In the nervous tissue and muscles of mammals, acetylcholinesterase (AChE,2 EC 3.1.1.7) controls cholinergic transmission by rapidly hydrolyzing the neurotransmitter acetylcholine after its release from nerve terminals
We have shown that a significant fraction of AChE catalytic subunits of type T (AChET) subunits is degraded intracellularly, through the ERAD process (“endoplasmic reticulumassociated degradation”) [20, 21], and that this is mostly induced by exposed aromatic residues [21, 22], in agreement with the fact that the formation of a complex in which these residues are occluded may reduce their degradation and increase their secretion
By using deletions and mutations, we analyze the influence of residues flanking the polyproline stretches and of the leucine located between the prolines, and we define a peptidic motif (RP4LP10RL), which is nearly as efficient as the complete extracellular domain of PRiMA for recruitment of AChET subunits into secreted heteromeric complexes
Summary
The existence of an N-glycosylated 20-kDa hydrophobic protein, associated with membrane-bound AChE tetramers, was originally discovered in 1987 by Gennari et al [14] and by Inestrosa et al [15]; this membrane anchor was more recently cloned and called PRiMA (“proline-rich membrane anchor”), because it contains a proline-rich motif, like ColQ [16]. By using deletions and mutations, we analyze the influence of residues flanking the polyproline stretches and of the leucine located between the prolines, and we define a peptidic motif (RP4LP10RL), which is nearly as efficient as the complete extracellular domain of PRiMA for recruitment of AChET subunits into secreted heteromeric complexes
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