Tsao Y S, Ivessa N E, Adesnik M, Sabatini D D, Kreibich G
Department of Cell Biology, New York University School of Medicine, New York 10016.
J Cell Biol. 1992 Jan;116(1):57-67. doi: 10.1083/jcb.116.1.57.
Two COOH terminally truncated variants of ribophorin I (RI), a type I transmembrane glycoprotein of 583 amino acids that is segregated to the rough portions of the ER and is associated with the protein-translocating apparatus of this organelle, were expressed in permanent HeLa cell transformants. Both variants, one membrane anchored but lacking part of the cytoplasmic domain (RL467) and the other consisting of the luminal 332 NH2-terminal amino acids (RI332), were retained intracellularly but, in contrast to the endogenous long lived, full length ribophorin I (t 1/2 = 25 h), were rapidly degraded (t 1/2 less than 50 min) by a nonlysosomal mechanism. The absence of a measurable lag phase in the degradation of both truncated ribophorins indicates that their turnover begins in the ER itself. The degradation of RI467 was monophasic (t 1/2 = 50 min) but the rate of degradation of RI332 molecules increased about threefold approximately 50 min after their synthesis. Several pieces of evidence suggest that the increase in degradative rate is the consequence of the transport of RI332 molecules that are not degraded during the first phase to a second degradative compartment. Thus, when added immediately after labeling, ionophores that inhibit vesicular flow out of the ER, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin, suppressed the second phase of degradation of RI332. On the other hand, when CCCP was added after the second phase of degradation of RI332 was initiated, the degradation was unaffected. Moreover, in cells treated with brefeldin A the degradation of RI332 became monophasic, and took place with a half-life intermediate between those of the two normal phases. These results point to the existence of two subcellular compartments where abnormal ER proteins can be degraded. One is the ER itself and the second is a non-lysosomal pre-Golgi compartment to which ER proteins are transported by vesicular flow. A survey of the effects of a variety of other ionophores and protease inhibitors on the turnover of RI332 revealed that metalloproteases are involved in both phases of the turnover and that the maintenance of a high Ca2+ concentration is necessary for the degradation of the luminally truncated ribophorin.
核糖体结合蛋白I（RI）是一种含583个氨基酸的I型跨膜糖蛋白，定位于内质网（ER）的粗面部分，并与该细胞器的蛋白质转运装置相关。其两个COOH末端截短的变体在HeLa细胞永久转化体中表达。这两个变体，一个锚定在膜上但缺少部分胞质结构域（RL467），另一个由332个NH2末端的腔内氨基酸组成（RI332），都保留在细胞内，但与内源性长寿命的全长核糖体结合蛋白I（半衰期=25小时）不同，它们通过非溶酶体机制迅速降解（半衰期小于50分钟）。两种截短的核糖体结合蛋白在降解过程中均未观察到明显的延迟期，这表明它们的周转始于内质网本身。RI467的降解是单相的（半衰期=50分钟），但RI332分子在合成后约50分钟其降解速率增加了约三倍。几条证据表明，降解速率的增加是由于第一阶段未降解的RI332分子转运到第二个降解区室所致。因此，在标记后立即添加抑制内质网囊泡流出的离子载体，如羰基氰化物间氯苯腙（CCCP）和莫能菌素，可抑制RI332降解的第二阶段。另一方面，当在RI332降解的第二阶段开始后添加CCCP时，降解不受影响。此外，在用布雷菲德菌素A处理的细胞中，RI332的降解变为单相，其半衰期介于两个正常阶段之间。这些结果表明存在两个亚细胞区室，异常的内质网蛋白可在其中降解。一个是内质网本身，另一个是内质网蛋白通过囊泡流转运到的非溶酶体高尔基前区室。对多种其他离子载体和蛋白酶抑制剂对RI332周转的影响进行的调查显示，金属蛋白酶参与了周转的两个阶段，并且维持高Ca2+浓度对于腔内截短的核糖体结合蛋白的降解是必要的。
