McLaughlin R E, Denny J B
PPD Development, Austin, TX 78704, USA.
Biochim Biophys Acta. 1999 Aug 12;1451(1):82-92. doi: 10.1016/s0167-4889(99)00074-9.
Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation.
神经元可塑性蛋白GAP - 43的棕榈酰化先前已被证明发生在质膜上，但初始棕榈酰化的位点尚未确定。为了确定这个细胞器，我们将GAP - 43与各种亚细胞组分一起孵育，并通过Triton X - 114分配法分析棕榈酰化。体外翻译的[(35)S]甲硫氨酸标记的GAP - 43与质膜、细胞核、线粒体、高尔基体以及含有内质网-高尔基体中间区室（ERGIC）但不含有质膜或高尔基体的粗微粒体制剂一起孵育。在质膜、高尔基体和ERGIC膜存在的情况下，GAP - 43会分配到Triton X - 114中，但在细胞核或线粒体存在时则不会。ERGIC引起的分配会被用棕榈酰化抑制剂二硫苏糖醇、衣霉素和低温预处理膜，以及用碘乙酰胺处理GAP - 43所阻断。分配的时间进程与先前报道的放射性棕榈酸掺入蛋白质的时间进程密切一致。由于ERGIC在细胞中分布广泛，我们的结果提供了证据表明ERGIC是GAP - 43棕榈酰化的初始位点。
