Lysosomal Proenzyme Sorting: A New Receptor

到目前为止，一种未知的受体可能是蛋白酶靶向溶酶体的关键，它已经在这个实验室中被鉴定出来。溶酶体蛋白原L和D是小鼠成纤维细胞来源的微体囊泡在酸性pH条件下的膜结合蛋白。这种结合是PRO酶所特有的，但不是成熟酶所特有的，它是可饱和的，并由一种可以通过蛋白酶处理从膜上移除的因子介导，这表明这是一种与受体蛋白的特异性相互作用。由于这种结合发生在溶酶体酶从甘露糖-6-磷酸受体上释放出来的pH，膜结合可能参与前溶酶体中的蛋白质分选。根据L原蛋白的氨基末端序列合成的多肽将被用于结合分析，以确定L前蛋白的哪个区域与膜相关。受体结合区的特定氨基酸将通过定点突变而改变。这些分析将确定负责正确的亚细胞靶向的特定氨基酸。改变后的重组蛋白将通过SV40启动子在COS细胞中表达，从细胞培养上清液中分离出来，并检测其膜结合能力。在这一初步筛选中，被确认为改变的蛋白已经失去了以依赖于pH的方式与膜结合的能力，随后将进行体内功能分析。该表达载体将在HeLa细胞中表达，证实能正确靶向野生型重组溶酶体酶。这些研究将确定负责pH依赖的膜结合的氨基酸拉伸是否包含溶酶体靶向所必需的信息。真核细胞的内部是高度分隔的，以便细胞执行其必要的功能。一组关键功能，包括内吞或自噬的大分子的水解性降解，被限制在被称为溶酶体的膜封闭的酸性隔间。这些降解是由蛋白质水解酶、糖苷酶和其他酸性水解酶催化的，这些酶包含在溶酶体中。这个项目解决了水解酶如何进入溶酶体的问题。几年前，人们发现许多溶酶体酶都有一个共同的寡糖结构，特别是甘露醇-6-磷酸残基，它被两个膜结合的MAN-6-P受体识别和结合。这些受体通过内酶体-溶酶体系统进行运输，并将结合的溶酶体酶传递到内酶体-溶酶体室。然而，越来越多的证据表明，MAN-6-P受体不能解释溶酶体酶在细胞中靶向的所有情况。因此，一种新的溶酶体酶结合膜蛋白的发现是一个重要的发现，它不是通过溶酶体酶上的man-6-P残基作用的，这无疑将拓宽我们对溶酶体酶如何进入溶酶体的看法。

A heretofore unidentified receptor which may be critical for protease targeting to lysosomes has been identified in this laboratory. Two lysosomal proproteases, procathepsins L and D, are membrane associated at acidic pH in microsomal vesicles derived from mouse fibroblasts. The binding, which is specific for the pro but not the mature enzymes, is saturable and is mediated by a factor which can be removed from the membranes by protease treatment, suggesting that this is a specific interaction with a receptor protein. As the association occurs at the pH at which lysosomal enzymes are released from mannose-6-phosphate receptors, the membrane association could be involved in protein sorting in prelysosomes. Synthetic peptides based on the amino-terminal sequence of procathepsin L will be used in binding assays to establish which region of the capthepsin L propeptide associates with membranes. Specific amino acids in the receptor binding regions will be altered by site-directed mutagenesis. These analyses will identify specific amino acids responsible for correct subcellular targeting. Altered recombinant protein will be expressed from an SV40 promoter in COS cells, isolated from cell culture medium, and assayed for membrane binding ability. Following this initial screen in which an altered protein is identified with has lost its ability to associate with membranes in a pH-dependent manner, an in vivo functional assay will be performed. The cDNA plasmid will be expressed in HeLa cells which have been demonstrated to target wild-type recombinant lysosomal enzymes correctly. These studies will establish whether the stretch of amino acids responsible for the pH-dependent membrane binding contains information essential for lysosomal targeting. %%% The interior of a eukaryotic cell is highly compartmentalized, in order for the cell to carry out its necessary functions. One set of critical functions, involving the hydrolytic degradation of endocytosed or autophagocytosed macromolecules, is restricted to a membrane-enclosed acidic compartment called the lysosome. These degradations are catalyzed by proteolytic, glycosidic, and other acid hydrolytic enzymes, which are contained within the lysosomes. This project addresses the question of how the hydrolytic enzymes get into the lysosomes. Some years ago, it was found that many lysosomal enzymes shared a common oligosaccharide structure, specifically a mannosyl-6-phosphate residue, which is recognized and bound by two membrane bound man-6-P receptors. These receptors traffic through the endosome-lysosome system and deliver bound lysosomal enzymes to the endosomal-lysosomal compartment. However, evidence has been accumulating in the literature that the man-6-P receptors cannot account for all instances of lysosomal enzyme targeting in cells. The discovery of a new lysosomal enzyme binding membrane protein, which does not act via the man-6-P residues on lysosomal enzymes, is therefore an important finding which will undoubtedly broaden our view of how lysosomal enzymes get into lysosomes.