Biology Department, University of Arkansas at Little Rock, 2801 South University Ave, Little Rock, AR 72204-1099, USA.
J Biosci. 2013 Sep;38(3):509-21. doi: 10.1007/s12038-013-9338-3.
Dictyostelium discoideum possesses only one caspase family member, paracaspase (pcp). Two separate mutant cell lines were first analysed: one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles. This association was disrupted when cells were exposed to osmotic stress conditions. Compared with wild-type cells, the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and were seen to be very rounded in hypo-osmotic conditions and contained more abnormally swollen contractile vacuole. Cells with pcp- were also rounded but had few, if any, contractile vacuoles. These observations suggest that Pcp is essential for Dictyostelium osmotic regulation via its functioning in the contractile vacuole system. Subjecting these cells to selected contractile vacuole inhibitor provided additional support for these findings. Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell.
粘菌盘基网柄菌只拥有一个胱冬肽酶家族成员，副半胱氨酸蛋白酶（pcp）。最初分析了两个独立的突变细胞系：一个细胞系是过表达 GFP 标记的 Pcp（GFP-Pcp），另一个细胞系是 pcp 缺失（pcp-）。表达 GFP-Pcp 的细胞的显微镜分析表明，Pcp 与由膀胱样囊泡组成的收缩泡膜相关。当细胞暴露于渗透胁迫条件时，这种关联被破坏。与野生型细胞相比，GFP-Pcp 过表达细胞对渗透胁迫敏感，在低渗条件下变得非常圆，并且含有更多异常肿胀的收缩泡。pcp-的细胞也呈圆形，但收缩泡很少，如果有的话。这些观察结果表明，Pcp 通过在收缩泡系统中的功能对粘菌盘基网柄菌的渗透调节至关重要。对这些细胞进行选定的收缩泡抑制剂处理为这些发现提供了额外的支持。此外，酵母双杂交系统鉴定出液泡质子 ATP 酶（VatM）作为与 Pcp 相互作用的蛋白质。总之，这项工作为真核副半胱氨酸蛋白酶与收缩泡系统的定位和调节相关提供了证据，收缩泡系统是维持细胞正常形态的关键细胞器。
