Komada M, Masaki R, Yamamoto A, Kitamura N
Institute for Liver Research, Kansai Medical University, Moriguchi, Osaka 570, Japan.
J Biol Chem. 1997 Aug 15;272(33):20538-44. doi: 10.1074/jbc.272.33.20538.
Hrs is a 115-kDa double zinc finger protein that is rapidly tyrosine phosphorylated in growth factor-stimulated cells. However, its function remains unknown. Here we show that Hrs is localized to early endosomes. Intracellular localization of endogenous Hrs and exogenously expressed Hrs tagged with the hemagglutinin epitope was examined by immunofluorescence staining using anti-Hrs and anti-hemagglutinin epitope antibodies, respectively. Hrs was detected in vesicular structures and was colocalized with the transferrin receptor, a marker for early endosomes, but only partially with CD63, a marker for late endosomes. A zinc finger domain deletion mutant of Hrs was also colocalized with the transferrin receptor, suggesting that the zinc finger domain is not required for its correct localization. Immunoelectron microscopy showed that Hrs was localized to the cytoplasmic surface of these structures. By subcellular fractionation, Hrs was recovered both in the cytoplasmic and membrane fractions. The membrane-associated Hrs was extracted from the membrane by alkali treatment, suggesting that it is peripherally associated with early endosomes. These results, together with our finding that Hrs is homologous to Vps27p, a protein essential for protein traffic through a prevacuolar compartment in yeast, suggest that Hrs is involved in vesicular transport through early endosomes.
Hrs是一种115 kDa的双锌指蛋白，在生长因子刺激的细胞中会迅速发生酪氨酸磷酸化。然而，其功能仍然未知。在此我们表明Hrs定位于早期内体。分别使用抗Hrs抗体和抗血凝素表位抗体，通过免疫荧光染色检测内源性Hrs和用血凝素表位标记的外源性表达Hrs的细胞内定位。在囊泡结构中检测到Hrs，它与早期内体的标志物转铁蛋白受体共定位，但仅部分地与晚期内体的标志物CD63共定位。Hrs的锌指结构域缺失突变体也与转铁蛋白受体共定位，这表明其正确定位不需要锌指结构域。免疫电子显微镜显示Hrs定位于这些结构的细胞质表面。通过亚细胞分级分离，在细胞质和膜级分中均回收了Hrs。膜相关的Hrs通过碱处理从膜中提取出来，这表明它与早期内体是外周相关的。这些结果，连同我们发现Hrs与Vps27p同源，Vps27p是酵母中通过前液泡区室进行蛋白质运输所必需的一种蛋白质，表明Hrs参与通过早期内体的囊泡运输。
