Analysis of the Membrane Structures Involved in Autophagy in Yeast by Freeze-Replica Method

Abstract

Under starvation conditions, the yeast S. cerevisiae sequesters its owncytoplasmic components by forming autophagosomes with double membrane in the cytoplasm. The autophagosome then fuses with the vacuolar membraneand delivers its owncytoplasmic componentsinto the vacuole in the form of an autophagic body with a single membrane (Baba, M., Takeshige, K., Baba, N., and Ohsumi, Y. 1994. J. Cell. Biol., 124: 903-913). Weexamined membrane structures involved in the autophagy induced by nitrogen-starvation by using freeze-replica method. The most conspicuous characteristic of the autophagic body is that the intramembrane particles were rarely detected on either the protoplasmic or exoplasmic face of its fracture membrane. This morphological feature of the fractured face was clearly different from other intracellular organelles. Next we examined the autophagosomal membrane. The inner membrane of autophagosome was also intramembrane particlefree, and its morphological feature was identical to the membrane of autophagic body. At the fusion site between autophagosome and vacuole we obtained direct evidence that two differnt membranes, the outer membrane of autophagosome and vacuolar membrane, became continuous by using freeze-etching technique. From these results we concluded that the autophagic body originated from the inner membrane of the autophagosome, and its membrane reflects an intrinsic feature of autophagosomal membrane. The outer membrane of autophagosome had only a few intramembrane particles and may be differentiated from the inner membrane. In cells under nitrogen-starvation condition, the density of intramembrane particles of vacuolar membrane decreased beyond that in control cells. © 1995, Japan Society for Cell Biology. All rights reserved.