Kreiner T, Moore H P
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Cell Regul. 1990 Apr;1(5):415-24. doi: 10.1091/mbc.1.5.415.
Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.
膜运输在细胞分裂过程中受到调控。具体而言，以病毒膜蛋白作为标记物，有研究表明有丝分裂细胞中内质网（ER）到高尔基体的运输基本受阻。然而，有丝分裂对分泌途径中其他步骤的影响尚不清楚，因为早期的运输阻断使得后续步骤的研究变得困难。在此，我们通过使用多种标记物，报告了对有丝分裂的哺乳动物组织培养细胞中分泌途径功能特性的研究。用编码分泌蛋白的cDNA转染中国仓鼠卵巢细胞。与早期使用病毒膜蛋白后的结果一致，我们发现有丝分裂细胞中的整体分泌途径无功能，分泌的主要阻断发生在内质网和高尔基体之间的步骤：有丝分裂细胞中人生长激素的整体分泌速率至少降低了10倍，并且通过获得内切糖苷酶H抗性判断，截短的水泡性口炎病毒G蛋白从内质网的输出受到了大致相同程度的抑制。为了确定从反式高尔基体到质膜的运输完整性，我们追踪了硫酸化糖胺聚糖（GAG）链的分泌，GAG链在高尔基体中合成，因此不受早期内质网到高尔基体阻断的影响。GAG链是组成型分泌蛋白所走途径的有效标记物；蛋白质分泌和GAG链分泌都对N-乙基马来酰亚胺和莫能菌素处理敏感，并在19℃下被阻断。我们发现，尽管与间期细胞相比，有丝分裂期间GAG链的初始分泌速率降低了约两倍，但有丝分裂期间GAG链的分泌程度并未改变。因此，在有丝分裂期间，从反式高尔基体到质膜的运输比内质网到高尔基体的运输受阻碍程度要小得多。我们得出结论，有丝分裂期间不同的运输步骤受到的影响程度不同。
