Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes.
Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes.
复制标题
重建高尔基体囊泡 CMP-唾液酸和腺苷 3-磷酸 5-磷酸硫酸盐转运至蛋白脂质体中。
DOI：
10.1073/pnas.86.6.1786
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发表时间：
1989
影响因子：
11.1
通讯作者：
Hirschberg,CB
中科院分区：
文献类型：
--
作者：
Milla,ME;Hirschberg,CB
我们以前已经证明高尔基体小泡将核苷酸糖和核苷酸硫酸盐运输到它们的管腔中。这些转运活动是细胞器和底物特有的，并以低微摩尔范围内核苷酸衍生物的表观Km为特征。作为我们纯化和鉴定上述转运蛋白的目标的一部分，我们已经将大鼠肝脏高尔基膜中的蛋白提取物重组为磷脂酰胆碱脂质体。由此产生的蛋白脂质体运输CMP-N-乙酰神经氨酸(CMP-AcNeu)和腺苷3‘-磷酸5’-磷酸硫酸盐，其亲和力和抑制特性与完整的高尔基体囊泡非常相似。唾液酸和硫酸钠只以非常缓慢的速度运输到高尔基体小泡的腔内，而重组的蛋白脂质体以低速率运输。无论是粗面内质网衍生的小泡，还是由粗面内质网蛋白质组成的脂质体，都不能运输cMP-AcNeu。以上结果表明，该重组系统可用于进一步纯化和鉴定核苷酸糖和核苷酸硫酸盐转运蛋白。这种方法对研究溶酶体和内体的膜运输蛋白也是有用的。
We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide sulfate into their lumen. These transport activities are organelle and substrate specific and are characterized by apparent Km for nucleotide derivatives in the low micromolar range. As part of our goal of purifying and characterizing the above transport proteins, we have reconstituted a protein extract from rat liver Golgi membranes into phosphatidylcholine liposomes. The resulting proteoliposomes transport CMP-N-acetylneuraminic acid (CMP-AcNeu) and adenosine 3'-phosphate 5'-phosphosulfate with very similar affinity and inhibition characteristics as intact Golgi vesicles. Sialic acid and sodium sulfate, which are transported only very slowly into the lumen of Golgi vesicles, are transported at low rates by the reconstituted proteoliposomes. Neither rough endoplasmic reticulum-derived vesicles nor proteoliposomes made from proteins of the rough endoplasmic reticulum transport CMP-AcNeu. The above results demonstrate that this reconstituted system can be used for further purification and characterization of nucleotide sugar and nucleotide sulfate translocator proteins. This approach should also be useful to study membrane transport proteins of lysosomes and endosomes.