Tavoularis S, Scazzocchio C, Sophianopoulou V
Institute of Biology, National Center for Scientific Research Demokritos (NCSRD), Aghia Paraskevi 153 10, Athens, Greece.
Fungal Genet Biol. 2001 Jul;33(2):115-25. doi: 10.1006/fgbi.2001.1280.
The PrnB protein is a highly specific proline transporter that belongs to an amino acid transporter family conserved in both prokaryotes and eukaryotes. In this work, we detected and analyzed the cellular localization of PrnB in vivo by means of green fluorescent protein (GFP) fusions. Several prnB-gfp gene fusions, driven by prnB native promoter sequences, were constructed and targeted to the genomic locus of a prnB null mutant. Chimeric proteins containing GFP fused to the C terminus of PrnB through a linker of two, four, or eight amino acids, with low potential to form secondary structure elements, were shown to be functional in vivo. A two-linker fusion results in partial complementation at both 25 and 37 degrees C. A four-linker fusion affords almost full complementation at 25 degrees C but partial complementation at 37 degrees C, whereas the eight-linker fusion results in partial complementation at both temperatures but in no GFP fluorescence. These results show that the number of linker amino acids is critical for the correct expression and/or translocation of PrnB-GFP fused proteins to the plasma membrane and for the fluorescence of the GFP. The expression of the four-linker PrnB-GFP transporter was detected and analyzed in vivo by both conventional fluorescence and confocal laser microscopy. This chimeric protein is localized in the plasma membrane, secondarily in large vacuoles found in the swollen conidial end of the germlings, and in other small intracellular structures observed as fluorescent granules. A strong correlation between known patterns of PrnB expression and intensity of PrnB-GFP fluorescence was observed. This work also demonstrates that the GFP fusion technology is a unique tool with which to study the expression and cellular localization of low-abundance transmembrane transporters expressed from their native promoters.
PrnB蛋白是一种高度特异性的脯氨酸转运蛋白，属于在原核生物和真核生物中都保守的氨基酸转运蛋白家族。在这项研究中，我们通过绿色荧光蛋白（GFP）融合技术在体内检测并分析了PrnB的细胞定位。构建了几个由PrnB天然启动子序列驱动的prnB - gfp基因融合体，并将其靶向到prnB基因敲除突变体的基因组位点。含有通过两个、四个或八个氨基酸的连接子与PrnB的C末端融合的GFP的嵌合蛋白，因其形成二级结构元件的可能性低，在体内被证明是有功能的。双连接子融合在25℃和37℃时都能实现部分互补。四连接子融合在25℃时几乎能完全互补，但在37℃时只能部分互补，而八连接子融合在两个温度下都只能部分互补，且没有GFP荧光。这些结果表明，连接子氨基酸的数量对于PrnB - GFP融合蛋白正确表达和/或转运到质膜以及GFP的荧光至关重要。通过传统荧光显微镜和共聚焦激光显微镜在体内检测并分析了四连接子PrnB - GFP转运蛋白的表达。这种嵌合蛋白定位于质膜，其次定位于在芽管膨大的分生孢子末端发现的大液泡，以及观察为荧光颗粒的其他小细胞内结构。观察到PrnB已知的表达模式与PrnB - GFP荧光强度之间有很强的相关性。这项研究还表明，GFP融合技术是研究从其天然启动子表达的低丰度跨膜转运蛋白的表达和细胞定位的独特工具。
