Park R D, Sullivan P C, Storrie B
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute, State University, Blacksburg 24061.
J Cell Physiol. 1988 Jun;135(3):443-50. doi: 10.1002/jcp.1041350311.
Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes sensitive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: 1) in vitro endosomes were osmotically insensitive; and 2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1-10 min at 37 degrees C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate-conjugated dextran (FITC-dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC-dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10-min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose ("very early" endosomes) and the other not ("early" endosomes). After a brief uptake (1-3 min), postincubation of CHO cells in 0.25 M sucrose-containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions.
将动物细胞与高渗蔗糖和聚乙二醇（PEG）1000一起孵育，可使内体在原位对低渗休克敏感（冈田和雷施泰纳，1982年）。我们发现：1）体外内体对渗透压不敏感；2）高渗蔗糖抑制从非常早期内体到溶酶体的转运。通过在37℃下将中国仓鼠卵巢（CHO）细胞与辣根过氧化物酶（HRP）和/或异硫氰酸荧光素偶联的葡聚糖（FITC-葡聚糖）孵育1 - 10分钟来标记内吞小泡。在0.25M蔗糖中制备的细胞组分通过用pH 6.7的5mM磷酸钠缓冲液稀释至最终蔗糖浓度为0.05M而受到低渗休克。低渗休克后，内化的HRP和FITC-葡聚糖与膜一起沉淀，而溶酶体水解酶则没有。沉淀中的HRP活性是潜伏性的，表明内体对渗透压休克有抗性。在高渗蔗糖存在下的摄取对内体随后的渗透压敏感性影响很小。在高渗蔗糖和PEG 1000存在下的摄取使内体对细胞匀浆敏感。出乎意料的是，在摄取和追踪培养基中加入高渗蔗糖会抑制溶酶体中HRP的出现。在10分钟摄取期间内化的HRP似乎存在于两个物理上不同的区室中，一个可被外源蔗糖抑制转运（“非常早期”内体），另一个则不能（“早期”内体）。短暂摄取（1 - 3分钟）后，将CHO细胞在含0.25M蔗糖的培养基中孵育可完全阻断内化的HRP向溶酶体的转运。这种阻断可通过将转化酶与HRP共同内化而部分缓解。这些结果表明，多个早期内体群体之间的转运对内细胞器内的渗透条件敏感。
