Browsing by Author "Voss, C."

Autophagy is the non-selective transport of proteins and superfluous organelles destined for degradation to the vacuole in fungae, or the lysosome in animal cells. Some of the genes encoding components of the autophagy pathway are conserved in plants, and here we show that Arabidopsis homologues of yeast Atg8 (Apg8/Aut7) and Atg4 (Apg4/Aut2) partially complement the yeast deletion strains. The yeast double mutant, a deletion strain with respect to both Atg8 and Atg4, could not be complemented by Arabidopsis Atg8, indicating that Arabidopsis Atg8 requires Atg4 for its function. Moreover, Arabidopsis Atg8 and Arabidopsis Atg4 interact directly in a two-hybrid assay. Interestingly, Atg8 shows significant homolgy with the microtubule binding light chain 3 of MAP1A and B, and here we show that Arabidopsis Atg8 hinds microtubuies. Our results demonstrate that a principle component of the autophagic pathway in plants is similar to that in yeast and we suggest that microtubule binding plays a role in this process. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Atg21 and Atg18 are homologue yeast proteins. Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway. Our proteinase protection experiments now demonstrate that growing atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21Delta cells showed only 35% of the wild-type rate. This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy. The intracellular localization of Atg21 is unique among the Atg proteins. In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions. Cells with a single vacuole show most of the protein at few perivacuolar punctae. This distribution pattern is reminiscent to the Vps class C( HOPS) ( homotypic fusion and vacuolar protein sorting) protein complex. In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure. Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded. Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors. However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.

The genome of the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) JaCP contains a cellular insertion coding for light chain 3 (LC3) of microtubule-associated proteins, the mammalian homologue of yeast Aut7p/Apg8p. The cellular insertion induces cp BVDV-specific processing of the viral polyprotein by a cellular cysteine protease homologous to the known yeast protease Aut2p/Apg4p. Three candidate bovine protease genes were identified on the basis of the sequence similarity of their products with the Saccharomyces cerevisiae enzyme. The search for a system for functional testing of these putative 1-0-specific proteases revealed that the components involved in this processing have been highly conserved during evolution, so that the substrate derived from a mammalian virus is processed in cells of mammalian, avian, fish, and insect origin, as well as in rabbit reticulocyte lysate, but not in wheat germ extracts. Moreover, two of these proteases and a homologous protein from chickens were able to rescue the defect of a yeast AUT2 deletion mutant. In coexpression experiments with yeast and wheat germ extracts one of the bovine proteases and the corresponding enzyme from chickens were able to process the viral polyprotein containing LC3. Northern blots showed that bovine viral diarrhea virus infection of cells has no significant influence on the expression of either LC3 or its protease, bAut2B2. However, LC3-specific processing of the viral polyprotein containing the cellular insertion is essential for replication of the virus since mutants with changes in the LC3 insertion significantly affecting processing at the LC3/NS3 site were not viable.

Autophagosomes and Cvt vesicles are limited by two membrane layers. The biogenesis of these unconventional vesicles and the origin of their membranes are hardly understood. Here we identify in Saccharomyces cerevisiae Trs85, a nonessential component of the TRAPP complexes, to be required for the biogenesis of Cvt vesicles. The TRAPP complexes function in endoplasmic reticulum-to-Golgi and Golgi trafficking. Growing trs85 Delta cells show a defect in the organization of the preautophagosomal structure. Although proaminopeptidase I is normally recruited to the preautophagosomal structure, the recruitment of green fluorescent protein-Atg8 depends on Trs85. Autophagy proceeds in the absence of Trs85, albeit at a reduced rate. Our electron microscopic analysis demonstrated that the reduced autophagic rate of trs85 Delta cells does not result from a reduced size of the autophagosomes. Growing and starved cells lacking Trs85 did not show defects in vacuolar biogenesis; mature vacuolar proteinase B and carboxypeptidase Y were present. Also vacuolar acidification was normal in these cells. It is known that mutations impairing the integrity of the ER or Golgi block both autophagy and the Cvt pathway. But the phenotypes of trs85 Delta cells show striking differences to those seen in mutants with defects in the early secretory pathway. This suggests that Trs85 might play a direct role in the Cvt pathway and autophagy.