Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
Mol Biol Cell. 2011 Jun 1;22(11):1887-95. doi: 10.1091/mbc.E10-11-0924. Epub 2011 Apr 6.
Intracellular palmitoylation dynamics are regulated by a large family of DHHC (Asp-His-His-Cys) palmitoyl transferases. The majority of DHHC proteins associate with endoplasmic reticulum (ER) or Golgi membranes, but an interesting exception is DHHC2, which localizes to dendritic vesicles of unknown origin in neurons, where it regulates dynamic palmitoylation of PSD95. Dendritic targeting of newly synthesized PSD95 is likely preceded by palmitoylation on Golgi membranes by DHHC3 and/or DHHC15. The precise intracellular distribution of DHHC2 is presently unclear, and there is very little known in general about how DHHC proteins achieve their respective localizations. In this study, membrane targeting of DHHC2 in live and fixed neuroendocrine cells was investigated and mutational analysis employed to define regions of DHHC2 that regulate targeting. We report that DHHC2 associates with the plasma membrane, Rab11-positive recycling endosomes, and vesicular structures. Plasma membrane integration of DHHC2 was confirmed by labeling of an extrafacial HA epitope in nonpermeabilized cells. Antibody-uptake experiments suggested that DHHC2 traffics between the plasma membrane and intracellular membranes. This dynamic localization was confirmed using fluorescence recovery after photo-bleaching analysis, which revealed constitutive refilling of the recycling endosome (RE) pool of DHHC2. The cytoplasmic C-terminus of DHHC2 regulates membrane targeting and a mutant lacking this domain was associated with the ER. Although DHHC2 is closely related to DHHC15, these proteins populate distinct membrane compartments. Construction of chimeric DHHC2/DHHC15 proteins revealed that this difference in localization is a consequence of divergent sequences within their C-terminal tails. This study is the first to highlight dynamic cycling of a mammalian DHHC protein between clearly defined membrane compartments, and to identify domains that specify membrane targeting of this protein family.
细胞内棕榈酰化动力学受大量 DHHC（天冬氨酸-组氨酸-组氨酸-半胱氨酸）棕榈酰转移酶家族调控。大多数 DHHC 蛋白与内质网 (ER) 或高尔基体膜相关，但一个有趣的例外是 DHHC2，它定位于神经元中未知来源的树突小泡，在那里它调节 PSD95 的动态棕榈酰化。新合成的 PSD95 的树突靶向可能先于 DHHC3 和/或 DHHC15 在高尔基体膜上进行棕榈酰化。DHHC2 的精确细胞内分布目前尚不清楚，一般来说，DHHC 蛋白如何实现其各自的定位知之甚少。在这项研究中，研究了活细胞和固定神经内分泌细胞中 DHHC2 的膜靶向，并进行了突变分析以确定调节靶向的 DHHC2 区域。我们报告 DHHC2 与质膜、Rab11 阳性再循环内体和囊泡结构相关联。DHHC2 与质膜的整合通过在非渗透性细胞中标记额外的 HA 表位得到证实。抗体摄取实验表明 DHHC2 在质膜和细胞内膜之间运输。使用荧光恢复后光漂白分析证实了这种动态定位，该分析显示 DHHC2 的再循环内体 (RE) 池持续填充。DHHC2 的细胞质 C 末端调节膜靶向，缺乏该结构域的突变体与内质网相关联。尽管 DHHC2 与 DHHC15 密切相关，但这些蛋白定位于不同的膜隔室。构建 DHHC2/DHHC15 嵌合蛋白表明，这种定位差异是其 C 末端尾部内的差异序列的结果。这项研究首次强调了一种哺乳动物 DHHC 蛋白在明确界定的膜隔室之间的动态循环，并确定了指定该蛋白家族膜靶向的结构域。
