Hrs, a tyrosine kinase substrate with a conserved double zinc finger domain, is localized to the cytoplasmic surface of early endosomes.
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.
Komada
M
M
Institute for Liver Research, Kansai Medical University, Moriguchi, Osaka 570, Japan.
Masaki
R
R
Yamamoto
A
A
Kitamura
N
N
D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000737:chemistry; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry* / Q000502:physiology; D016335:Zinc Fingers
D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate