Bannan Barbra A, Van Etten Jamie, Kohler John A, Tsoi Yui, Hansen Nicole M, Sigmon Stacey, Fowler Elizabeth, Buff Haley, Williams Tiffany S, Ault Jeffrey G, Glaser Robert L, Korey Christopher A
Department of Biology; College of Charleston; Charleston, South Carolina, USA.
Fly (Austin). 2008 Jul-Aug;2(4):198-214. doi: 10.4161/fly.6621.
Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization,RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627 and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2)and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation.
棕榈酰化是指通过共价硫酯键将棕榈酸部分翻译后添加到半胱氨酸残基上的过程。这种修饰的添加和去除由棕榈酰酰基转移酶和硫酯酶共同控制。通过生物信息学分析，我们在果蝇基因组中鉴定出22个DHHC家族棕榈酰酰基转移酶同源物。我们使用原位杂交、逆转录聚合酶链反应（RT-PCR）以及已发表的果蝇图谱微阵列数据来表征所有22个果蝇同源物的表达模式。我们的结果表明，所有这些都是表达基因，但其中几个，包括CG1407、CG4676、CG5620、CG6017/dHIP14、CG6618、CG6627和CG17257，似乎在神经组织中富集，这表明它们对神经功能很重要。此外，我们发现其中几个可能以性别特异性方式表达，CG4483和CG17195在成年雄性中特异性表达。使用带有标签的DHHC基因版本，我们证明果蝇DHHC蛋白主要位于S2细胞中的高尔基体或内质网中，除了在质膜上发现的CG1407。我们还表征了三种已知硫酯酶的亚细胞定位和表达：棕榈酰蛋白硫酯酶1（Ppt1）、棕榈酰蛋白硫酯酶2（Ppt2）和酰基蛋白硫酯酶1（APT1）。我们的结果表明，Ppt1和Ppt2是主要的溶酶体硫酯酶，而APT1可能是细胞质硫酯酶。最后，体内拯救实验表明，Ppt2的表达不能挽救与Ppt1缺失相关的神经包涵体表型，进一步支持了这两种硫酯酶具有不同的功能和底物。这些结果将为更全面地了解果蝇中蛋白质棕榈酰化组的正常细胞功能奠定基础，并将有助于进一步深入了解与棕榈酰化失调相关疾病的分子病因。
