Brickman M J, Balber A E
Department of Immunology, Duke University Medical Center, Durham NC 27710.
J Cell Sci. 1994 Nov;107 ( Pt 11):3191-200. doi: 10.1242/jcs.107.11.3191.
gp57/42 is a membrane glycoprotein localized in the trans-Golgi, flagellar pocket region of the cell surface, endosomes and lysosomes of bloodstream forms of Trypanosoma brucei rhodesiense. Pulse-chase immunoprecipitation experiments revealed that gp57/42 acquires a unique N-linked oligosaccharide recognized by the CB1 monoclonal antibody 20-30 minutes after protein synthesis, probably in the trans-Golgi. We refer to gp57/42 molecules that carry the CB1 epitope as CB1-gp. Pulse labeled CB1-gp contained only one core protein, p57, when chase times were 30 minutes or less. As time of chase increased from 30 to 60 minutes, a new polypeptide, p42, appeared in N-glycanase-treated CB1 immunoprecipitates. Since p57 and p42 share 10 of 13 methionyl peptides, we conclude that p42 is a fragment of p57. Cleavage of p57 to p42 was not inhibited when cells were chased in two thiol protease inhibitors or in 3,4-diisocoumarin, but was inhibited by leupeptin. Cell surface biotinylation was used to determine if newly synthesized CB1-gp was transported from the Golgi to the surface. When cells were pulse labeled and chased for 30 minutes, as much as 40% of the radiolabeled CB1-gp could be biotinylated on the cell surface. The amount of CB1-gp that could be biotinylated decreased when chases were extended from 30 to 60 minutes, suggesting that pulse labeled CB1-gp left the surface. In contrast, pulse labeled variant surface glycoprotein molecules continued to accumulate on the surface where they could be biotinylated between 30 and 60 minutes of chase. Biotinylated CB1-gp derived from cells chased for 30 minutes contained p57 but no p42. However, when labeled cells were biotinylated after a 30 minute chase and then incubated another 30 minutes at 37 degrees C, the biotinylated CB1-gp contained both p57 and p42. The p57 in biotinylated CB1-gp was not cleaved to p42 if the additional incubation was done at 4 or 12 degrees C. This suggests that transport to a compartment where processing occurs and/or the processing enzymes are inhibited by low temperature. When surface biotinylation was done after a 60 minute chase, p42 was detected in biotinylated CB1-gp, suggesting that CB1-gp molecules had passed through the processing compartment and then appeared on the cell surface. Thus, a major portion of the newly synthesized CB1-gp is routed from the Golgi to endocytic compartments via the cell surface. In trypanosomes this process involves a unique surface domain, the flagellar pocket.(ABSTRACT TRUNCATED AT 400 WORDS)
gp57/42是一种膜糖蛋白，定位于布氏罗得西亚锥虫血流形式细胞的反式高尔基体、细胞表面鞭毛袋区域、内体和溶酶体中。脉冲追踪免疫沉淀实验表明，gp57/42在蛋白质合成后20 - 30分钟获得一种独特的N - 连接寡糖，可能是在反式高尔基体中，该寡糖可被CB1单克隆抗体识别。我们将携带CB1表位的gp57/42分子称为CB1 - gp。当追踪时间为30分钟或更短时间时，脉冲标记的CB1 - gp仅包含一种核心蛋白p57。随着追踪时间从30分钟增加到60分钟，一种新的多肽p42出现在用N - 糖苷酶处理的CB1免疫沉淀物中。由于p57和p42在13个甲硫氨酰肽中有10个相同，我们得出结论，p42是p57的片段。当细胞在两种巯基蛋白酶抑制剂或3,4 - 二异香豆素中进行追踪时，p57裂解为p42的过程未受抑制，但被亮抑蛋白酶肽抑制。细胞表面生物素化用于确定新合成的CB1 - gp是否从高尔基体转运到细胞表面。当细胞进行脉冲标记并追踪30分钟时，高达40%的放射性标记CB1 - gp可在细胞表面被生物素化。当追踪时间从30分钟延长到60分钟时，可被生物素化的CB1 - gp量减少，这表明脉冲标记的CB1 - gp离开了细胞表面。相比之下，脉冲标记的可变表面糖蛋白分子继续在细胞表面积累，在追踪30至60分钟之间它们可被生物素化。来自追踪30分钟的细胞的生物素化CB1 - gp包含p57但不包含p42。然而，当标记细胞在30分钟追踪后进行生物素化，然后在37℃再孵育30分钟时，生物素化的CB1 - gp同时包含p57和p42。如果在4℃或12℃进行额外孵育，生物素化CB1 - gp中的p57不会裂解为p42。这表明转运到一个发生加工的区室和/或加工酶被低温抑制。当在60分钟追踪后进行表面生物素化时，在生物素化的CB1 - gp中检测到p42，这表明CB1 - gp分子已经通过加工区室然后出现在细胞表面。因此，新合成的CB1 - gp的主要部分通过细胞表面从高尔基体被转运到内吞区室。在锥虫中，这个过程涉及一个独特的表面结构域，即鞭毛袋。（摘要截断于400字）
