Identification and characterization of COPla- and COPlb-type vesicle classes associated with plant and algal Golgi
Identification and characterization of COPla- and COPlb-type vesicle classes associated with plant and algal Golgi
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DOI：
10.1073/pnas.0609818104
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发表时间：
2007-01-02
影响因子：
11.1
通讯作者：
Staehelin, L. Andrew
中科院分区：
文献类型：
--
作者：
Donohoe, Bryon S.;Kang, Byung-Ho;Staehelin, L. Andrew
外壳蛋白 I (COPI) 囊泡起源于高尔基体池，介导蛋白质从高尔基体循环回到内质网 (ER) 以及高尔基体驻留蛋白在池之间的运输。体外研究已经为两种不同类型的 COPI 囊泡提供了证据，但这些囊泡的体内作用部位仍有待确定。我们结合使用电子断层扫描和免疫标记技术来检查通过高压冷冻/冷冻替代方法保存的鳞片产藻 Scherffelia dubia 和拟南芥细胞中的高尔基体堆栈和相关囊泡。区分了五种结构不同的囊泡类型。在拟南芥中，通过免疫金标记鉴定了 COPI 和 COPII 囊泡外壳蛋白以及囊泡货物分子（甘露糖苷酶 I 和唾液酸转移酶黄色荧光蛋白）。在这两种生物体中，COPI 型囊泡通过六个结构标准的组合进一步表征：外壳结构、外壳厚度、膜结构、货物染色、脑池起源和空间分布。使用这种多参数结构方法，我们可以区分两种类型的 COPI 囊泡：COPIa 和 COPIb。 COPIa 囊泡仅从顺式池中出芽，并占据顺式池和 ER 输出位点之间的空间，而 COPIb 囊泡仅从内侧高尔基体池和反式高尔基体池中出芽，并局限于这些后者池周围的空间。我们的结论是，COPIa 囊泡介导的内质网再循环仅发生在顺式池，COPIb 囊泡对高尔基体驻留蛋白的逆行转运仅限于内侧和反式池，并且高尔基体周围囊泡的扩散受到限制。
Coat protein I (COPI) vesicles arise from Golgi cisternae and mediate the recycling of proteins from the Golgi back to the endoplasmic reticulum (ER) and the transport of Golgi resident proteins between cisternae. In vitro studies have produced evidence for two distinct types of COPI vesicles, but the in vivo sites of operation of these vesicles remain to be established. We have used a combination of electron tomography and immunolabeling techniques to examine Golgi stacks and associated vesicles in the cells of the scale-producing alga Scherffelia dubia and Arabidopsis preserved by high-pressure freezing/freeze-substitution methods. Five structurally distinct types of vesicles were distinguished. In Arabidopsis, COPI and COPII vesicle coat proteins as well as vesicle cargo molecules (mannosidase I and sialyltransferase-yellow fluorescent protein) were identified by immunogold labeling. In both organisms, the COPI-type vesicles were further characterized by a combination of six structural criteria: coat architecture, coat thickness, membrane structure, cargo staining, cisternal origin, and spatial distribution. Using this multiparameter structural approach, we can distinguish two types of COPI vesicles, COPIa and COPIb. COPIa vesicles bud exclusively from cis cisternae and occupy the space between cis cisternae and ER export sites, whereas the COPIb vesicles bud exclusively from medial- and trans-Golgi cisternae and are confined to the space around these latter cisternae. We conclude that COPIa vesicle-mediated recycling to the ER occurs only from cis cisternae, that retrograde transport of Golgi resident proteins by COPIb vesicles is limited to medial and trans cisternae, and that diffusion of periGolgi vesicles is restricted.