Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation.
Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation.
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DOI：
10.1371/journal.pone.0064514
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Robinson MS
中科院分区：
文献类型：
--
作者：
Sahlender DA;Kozik P;Miller SE;Peden AA;Robinson MS
CALM（网格蛋白组装淋巴样髓性白血病蛋白）是后高尔基体R-SNARE VAMP 2、3和8的货物选择性适配器，并且它还调节质膜上网格蛋白包被的小凹和囊泡的大小。本研究有两个目标：确定CALM是否可以排序额外的VAMP，并调查VAMP排序是否有助于CALM依赖的囊泡大小调节。使用基于流式细胞术的内吞效率测定，我们证明CALM也能够分选VAMP 4和7，即使它们具有针对其他网格蛋白衔接子的分选信号。CALM同源物存在于几乎每一种真核生物中，这表明CALM家族可能已经进化为从质膜检索所有高尔基体后VAMP的适配器。使用敲除/救援系统，我们表明，野生型CALM恢复正常的VAMP分选CALM耗尽的细胞，但两个非VAMP结合突变体不。然而，当我们使用基于荧光显微镜的测定来测定CALM消耗对包被凹坑形态的影响时，我们发现两种突变体与野生型CALM一样有效。因此，我们可以将CALM的排序功能与其结构角色分离开来。
CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.
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影响因子：
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64.8
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