Mammalian brain-specific L-proline transporter. Neuronal localization of mRNA and enrichment of transporter protein in synaptic plasma membranes.
Mammalian brain-specific L-proline transporter. Neuronal localization of mRNA and enrichment of transporter protein in synaptic plasma membranes.
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哺乳动物脑特异性 L-脯氨酸转运蛋白。
DOI：
10.1074/jbc.270.26.15755
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
FremeauJr,RT
中科院分区：
文献类型：
--
作者：
Velaz-Faircloth,M;Guadaño-Ferraz,A;Henzi,VA;FremeauJr,RT
高亲和力Na+（和Cl+）依赖性L-脯氨酸转运蛋白（PROT）在大鼠脑中推定的谷氨酸能途径的亚群中的表达提高了该载体在兴奋性神经传递中的特定生理作用的可能性（Fremeau，R. T.，小的，卡伦，M. G.，和Blakely，R. D.（1992）Neuron 8，915-926）。然而，PROT蛋白的生化特性和区域、细胞和亚细胞分布尚未阐明。在这里，我们记录了大鼠PROT mRNA的脑特异性表达和神经元定位。我们还报告了哺乳动物脑PROT蛋白的第一个鉴定和部分生化特性。亲和纯化的抗肽抗体产生，特异性地识别一个单一的68-kDa的PROT蛋白对大鼠和人脑组织的免疫印迹。用肽-N-糖苷酶F对大鼠海马膜进行去糖基化，使原生PROT蛋白的表观分子量从68 kDa降低到53 kDa，这是在没有微粒体的情况下通过大鼠PROT cDNA的体外翻译确定的主要PROT翻译产物的大小。亚细胞分级分离研究表明，PROT蛋白是丰富的突触质膜，但缺乏突触后密度。在大鼠纹状体中观察到PROT mRNA和蛋白的差异分布，表明转运蛋白在皮质中的神经元细胞体中合成，并输出到尾壳核中的轴突终末。这些研究结果值得考虑一种新的突触前调节作用，这种转运兴奋性突触传递。
The expression of a high affinity Na⁺- (and Cl⁻) dependent L-proline transporter (PROT) in subpopulations of putative glutamatergic pathways in rat brain raises the possibility of a specific physiological role(s) for this carrier in excitatory neurotransmission (Fremeau, R. T., Jr., Caron, M. G., and Blakely, R. D.(1992)Neuron8, 915-926). However, the biochemical properties and regional, cellular, and subcellular distribution of the PROT protein have yet to be elucidated. Here, we document the brain-specific expression and neuronal localization of rat PROT mRNA. We also report the first identification and partial biochemical characterization of the mammalian brain PROT protein. An affinity-purified antipeptide antibody was produced that specifically recognized a single 68-kDa PROT protein on immunoblots of rat and human brain tissues. Deglycosylation of rat hippocampal membranes with peptide-N-glycosidase F reduced the apparent molecular mass of the native PROT protein from 68 to 53 kDa, the size of the primary PROT translation product determined byin vitrotranslation of the rat PROT cDNA in the absence of microsomes. Subcellular fractionation studies demonstrated that the PROT protein was enriched in synaptic plasma membranes but absent from postsynaptic densities. A differential distribution of PROT mRNA and protein was observed in rat striatum, suggesting that the transporter protein is synthesized in neuronal cell bodies in the cortex and exported to axon terminals in the caudate putamen. These findings warrant the consideration of a novel presynaptic regulatory role for this transporter in excitatory synaptic transmission.