Rab7：溶酶体生物发生的关键。,Molecular Biology of the Cell

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Rab7：溶酶体生物发生的关键。
Molecular Biology of the Cell ( IF 3.1 ) Pub Date : 2000-02-26 , DOI: 10.1091/mbc.11.2.467
C Bucci ₁ , P Thomsen , P Nicoziani , J McCarthy , B van Deurs

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溶酶体生物发生背后的分子机制以及晚期内吞结构的核周聚集体的维持尚不清楚。可能是该机制的一部分的候选蛋白是小GTPase Rab7，但尚不清楚该蛋白是否与溶酶体相关或在核周溶酶体区室的调节中起任何作用。以前，Rab7主要涉及从早期到晚期内体的转运。现在，我们已经使用一种新方法来分析Rab7的作用：在HeLa细胞中瞬时表达增强型绿色荧光蛋白（EGFP）标记的Rab7 wt和突变蛋白。EGFP-Rab7 wt与晚期胞吞结构相关，主要是溶酶体，其在核周区域聚集并融合。各个溶酶体的大小以及核周聚集的程度随EGFP-Rab7 wt的表达水平以及活性EGFP-Rab7Q67L突变体的表达水平而增加。相反，在主要定位于细胞质的显性阴性突变体EGFP-Rab7T22N和EGFP-Rab7N125I表达后，核周溶酶体聚集体消失，溶酶体（通过组织蛋白酶D和溶酶体相关膜蛋白-1的共定位而鉴定）变成了溶酶体。分散在整个细胞质中的内吞分子（如低密度脂蛋白）难以接近它们，并且其酸度大大降低，这是由酸性探针LysoTracker Red的堆积减少所确定的。相反，与Rab5和转铁蛋白受体相关的早期内体，富含不依赖阳离子的甘露糖6-磷酸受体的晚期内体和通过在TGN-38中富集而确定的反式高尔基网络不变。这些数据首次证明，控制晚期内吞结构/溶酶体的聚集和融合的Rab7对于维持核周溶酶体区室至关重要。

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Rab7: a key to lysosome biogenesis.

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.

更新日期：2019-11-01

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