A 60 kDa plasma membrane protein changes its localization to autophagosomeand autolysosome membranes during induction of autophagy in rat hepatoma cell line, H-4-II-E cells
Y. Tagawa et al., A 60 kDa plasma membrane protein changes its localization to autophagosomeand autolysosome membranes during induction of autophagy in rat hepatoma cell line, H-4-II-E cells, CELL STRUCT, 24(2), 1999, pp. 59-70
We previously reported the preparation and characterization of an antibody
against membrane fraction of autolysosomes from rat liver (J. Histochem. Cy
tochem. 38, 1571-1581, 1990). Immunoblot analyses of total membrane fractio
n of a rat hepatoma cell line, H-4-II-E cells by this antibody suggested th
at H-4-II-E cells expressed several autolysosomal proteins, including a pro
tein with apparent molecular weight of 60 kDa, It was suggested that this 6
0 kDa protein was a peripheral membrane protein, because it was eluted from
the membrane by sodium carbonate treatment. We prepared an antibody agains
t this 60 kDa protein by affinity purification method, and examined its beh
avior during induction of autophagy. Autophagy was induced by transferring
the cells from Dulbecco's modified Eagle medium (DMEM) containing 12% fetal
calf serum into Hanks' balance salt solution. In DMEM, the 60 kDa protein
showed diffused immunofluorescence pattern, and immunoelectron microscopy s
uggested that this protein was located on the extracellular side of the pla
sma membrane. After inducing autophagy, the immunofluorescence configuratio
n of the 60 kDa protein changed from the diffused pattern to a granulous on
e. Immunoelectron microscopy showed that the 60 kDa protein was localized o
n the luminal side of the limiting membrane of autolysosomes and endosomes,
In the presence of bafilomycin A(1) which prevents fusion between autophag
osomes and lysosomes, the 60 kDa protein was localized on the limiting memb
rane of the autophagosomes and endosomes, These results suggest that the 60
kDa protein is transported from the plasma membrane to the autophagosome m
embrane through the endosomes.