van Ooij Christiaan, Losick Richard
The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Bacteriol. 2003 Feb;185(4):1391-8. doi: 10.1128/JB.185.4.1391-1398.2003.
SpoVM is an unusually small (26-residue-long) protein that is produced in the mother cell chamber of the sporangium during the process of sporulation in Bacillus subtilis. We investigated the subcellular localization of SpoVM, which is believed to be an amphipathic alpha-helix, by using a fusion of the sporulation protein to the green fluorescence protein (GFP). We found that SpoVM-GFP is recruited to the polar septum shortly after the sporangium undergoes asymmetric division and that the fusion protein localizes to the mother cell membrane that surrounds the forespore during the subsequent process of engulfment. We identified a patch of three residues near the N terminus of the proposed alpha-helix that is needed both for proper subcellular localization and for SpoVM function. We also identified a patch of residues on the opposite face of the helix and residues near both ends of the protein that are needed for SpoVM function but not for subcellular localization. Subcellular localization of SpoVM-GFP was found to require an unknown gene(s) under the control of the mother cell transcription factor sigmaE. We propose that the N-terminal patch binds to an unknown anchoring protein that is produced under the control of sigmaE and that other residues important in SpoVM function to recruit an unknown sporulation protein(s) to the mother cell membrane that surrounds the forespore. Our results provide evidence that SpoVM function depends on proper subcellular localization.
SpoVM是一种异常小的（仅26个氨基酸长）蛋白质，它在枯草芽孢杆菌孢子形成过程中于芽孢囊的母细胞腔室中产生。我们通过将孢子形成蛋白与绿色荧光蛋白（GFP）融合，研究了被认为是两亲性α-螺旋的SpoVM的亚细胞定位。我们发现，在芽孢囊进行不对称分裂后不久，SpoVM-GFP就被募集到极隔膜处，并且在随后的吞噬过程中，融合蛋白定位于围绕前芽孢的母细胞膜上。我们在假定的α-螺旋N端附近鉴定出三个氨基酸组成的片段，该片段对于正确的亚细胞定位和SpoVM功能都是必需的。我们还在螺旋的相对面上鉴定出一片氨基酸残基以及蛋白质两端附近的残基，这些残基对于SpoVM功能是必需的，但对于亚细胞定位并非必需。发现SpoVM-GFP的亚细胞定位需要在母细胞转录因子sigmaE控制下的一个未知基因。我们提出，N端片段与在sigmaE控制下产生的一种未知锚定蛋白结合，并且SpoVM功能中其他重要残基可将一种未知的孢子形成蛋白募集到围绕前芽孢的母细胞膜上。我们的结果提供了证据，证明SpoVM的功能依赖于正确的亚细胞定位。
