Brand S H, Holtzman E J, Scher D A, Ausiello D A, Stow J L
Department of Medicine, Massachusetts General Hospital, Charlestown, USA.
Am J Physiol. 1996 May;270(5 Pt 1):C1362-9. doi: 10.1152/ajpcell.1996.270.5.C1362.
Heterotrimeric G protein alpha-subunits localized on the cytoplasmic face of Golgi membranes are involved in regulating vesicle trafficking and protein secretion. We investigated the role of myristoylation in attachment of the G alpha i-3 subunit to Golgi membranes. G alpha i-3 was epitope-tagged by insertion of a FLAG sequence at an NH2-terminal site predicted to interfere with myristoylation, and the resulting NT-alpha i-3 construct was stably transfected and expressed in polarized epithelial LLC-PK1 cells. Metabolic labeling confirmed that the translation product of NT-alpha i-3 was not myristoylated. In contrast to endogenous G alpha 1-3, which is tightly bound to Golgi membranes, the unmyristoylated FLAG-tagged NT-alpha i-3 did not attach to membranes; it was localized by immunofluorescence in the cytoplasm of LLC-PK1 cells and was detected only in the cytosol fraction of cell homogenates. Pertussis toxin-dependent ADP-ribosylation was used to test the ability of NT-alpha i-3 to interact with membrane-bound beta gamma-subunits. In both in vitro and in vivo assays, cytosolic NT-alpha i-3 alone was not ADP-ribosylated, although in the presence of membranes it could interact with G beta gamma-subunits to form heterotrimers. The expression of NT-alpha i-3 in LLC-PK1 cells altered the rate of basolateral secretion of sulfated proteoglycans, consistent with the demonstrated function of endogenous G alpha i-3. These data are consistent with a model in which G alpha i-3 utilizes NH2-terminal myristoylation to bind to Golgi membranes and to maximize its interaction with G beta gamma-subunits. Furthermore, our results show that stable attachment of G alpha i-3 to Golgi membranes is not required for it to participate as a regulatory element in vesicle trafficking in the secretory pathway.
定位于高尔基体膜胞质面的异源三聚体G蛋白α亚基参与调节囊泡运输和蛋白质分泌。我们研究了肉豆蔻酰化在Gαi - 3亚基与高尔基体膜附着中的作用。通过在预测会干扰肉豆蔻酰化的NH2末端位点插入FLAG序列对Gαi - 3进行表位标记，然后将所得的NT - αi - 3构建体稳定转染并在极化上皮LLC - PK1细胞中表达。代谢标记证实NT - αi - 3的翻译产物未被肉豆蔻酰化。与紧密结合于高尔基体膜的内源性Gα1 - 3不同，未肉豆蔻酰化的FLAG标记的NT - αi - 3不附着于膜；通过免疫荧光定位在LLC - PK1细胞的细胞质中，并且仅在细胞匀浆的胞质溶胶部分中检测到。利用百日咳毒素依赖性ADP核糖基化来测试NT - αi - 3与膜结合的βγ亚基相互作用的能力。在体外和体内试验中，单独的胞质溶胶NT - αi - 3都未被ADP核糖基化，尽管在有膜存在的情况下它可以与Gβγ亚基相互作用形成异源三聚体。NT - αi - 3在LLC - PK1细胞中的表达改变了硫酸化蛋白聚糖的基底外侧分泌速率，这与内源性Gαi - 3已证实的功能一致。这些数据与一个模型相符，即Gαi - 3利用NH2末端肉豆蔻酰化与高尔基体膜结合并最大化其与Gβγ亚基的相互作用。此外，我们的结果表明Gαi - 3稳定附着于高尔基体膜并非其作为分泌途径中囊泡运输调节元件参与所必需的。
