A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.
A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.
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无细胞测定允许重建 Vps33p 依赖性运输至酵母液泡/溶酶体。
DOI：
10.1083/jcb.146.1.85
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Gerhardt,B
中科院分区：
文献类型：
--
作者：
Vida,T;Gerhardt,B
我们报告了一个无细胞系统，它测量了羧基肽酶Y(CPY)的运输耦合成熟度。酵母球形体通过聚碳酸酯过滤器挤压裂解。经过差速离心后，125,000粒被浓缩为放射性标记的proCPY，并被用作“供体”膜。从非放射性标记细胞中提取的15,000粒浓缩的液泡被用作“受体”膜。当这些膜与三磷酸腺苷和胞浆提取物一起孵育时，放射性标记的前CPY的∼50%被加工成成熟的CPY。在孵化过程中，供体和受体膜的稀释抑制了成熟，表现出15min的滞后期，并且对温度敏感。当供体膜来自PEP4基因缺失的酵母菌株(编码主要的CPY加工酶，蛋白酶A)时，有效的前CPY成熟是可能的，而受体膜来自PEP4酵母菌株，这表明间隔室转移。由缺失VPS33基因的酵母菌株制成的胞浆在驱动运输方面效率较低。此外，针对Vps33p(一种Sec1同源物)和Vam3p(一种Q-SNARE)的抗体可以抑制90%的转运。高表达Vps33p酵母细胞的胞液提取物恢复了对抗体抑制检测的转运。这种无细胞系统使得重组的室间转运与VPS基因产物的功能耦合成为可能。
We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-gpellet is enriched for radiolabeled proCPY and is used as “donor” membranes. A 15,000-gpellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as “acceptor” membranes. When these membranes are incubated together with ATP and cytosolic extracts, ∼50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for thePEP4gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from aPEP4yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for theVPS33gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of aVPSgene product.