| dc.description.abstracteng | Autophagy is a ubiquitous protein
degradation pathway in eukaryotic cells. As response upon nutrient
starvation, cytosolic material and also whole organelles are
engulfed by a double membrane-layered vesicle, the autophagosome.
The formation of the autophagosome starts at the pre-autophagosomal
structure (PAS). The completed autophagosome is then targeted to
the vacuole. After fusion with the vacuole, a single
membrane-layered vesicle, the autophagic body, is released into the
vacuole. After lysis of the membrane, the contents of the
autophagic body are degraded and the contained macromolecules are
recycled. The aim of this study was the identification of
cooperating or redundant genes to different autophagic genes, to
uncover molecular details of autophagosome formation and to allow
mechanistic insights into different selective variants of
autophagy. For the identification of autophagy related cooperating
or redundant genes, the Synthetic Genetic Array analysis (SGA) was
used. This is a powerful high-throughput technique to identify
synthetic lethal double deletions, probably reflecting the same
essential function of the respective gene products. For this
purpose, the query strains atg8∆, atg15∆, atg18∆ and atg21∆ were
generated in this study and crossed to the entire deletion library
available for S. cerevisiae. Unfortunately, the procedure resulted
in only false positive matches.
In addition to the common marker protein GFP-Atg8, this study
introduces the marker protein Pgk1-GFP to detect unselective
autophagy by western blot and microscopy. Compared to the
autophagic substrate GFP-Atg8, the use of this cytosolic protein
allows the unbiased detection of unselective bulk autophagy.
The molecular details of the biogenesis of autophagosomes are
poorly understood. Our group identified the AAA+ ATPase Cdc48 and
its cofactor Ubx1 (Shp1) as novel components needed for
autophagosome biogenesis. In accordance with former experiments in
the laboratory, this study presents Shp1 as the exclusive cofactor
of Cdc48 needed for autophagy. SHP1 deleted cells show no
accumulation of autophagosomes in the vacuole, while the formation
of the PAS or the lipidation of Atg8, which both are important
steps during autophagosome formation, are not impaired in shp1∆
cells. Furthermore, a suggested interaction of Shp1 and Atg8 was
proven by recombinant pull down experiments using GST-Shp1 and some
truncated versions. In mammals, the homologues of Cdc48 and Shp1,
p97 and p47, are involved in the ubiquitin-dependent mitotic Golgi
reassembly. In contrast, the complex of Cdc48 and Shp1 in yeast,
requires the ubiquitin-like Atg8 for its function in autophagy.
This gives for the first time a rationale for the use of Atg8
during unselective autophagy and explains why the delipidation of
Atg8-PE by Atg4 is necessary for efficient autophagy.
Mitochondria are the sites of ATP production in aerobic cells. As a
consequence, they are at the same time a major source for reactive
oxygen species (ROS), which damage mitochondria and other
organelles. Thus, a quality control for mitochondria is essential
for cellular survival. Mitophagy, the selective autophagic
degradation of mitochondria, might fulfil such a role, but is still
poorly understood in yeast. In this study, the construction of a
marker protein and its use in degradation assay experiments by
western blot allow the quantitative measurement of mitophagy. In
contrast to a commonly used chromosomal Om45-GFP fusion, the novel
marker protein can be expressed vom a plasmid, avoiding time
consuming chromosomal integration. Different conditions to induce
mitophagy were established. These different conditions allow the
differentiation between mitophagy of superfluous or damaged
mitochondria. Hereby, proteins of the ESCRT machinery, not required
for unselective bulk autophagy, were found to be needed for
mitophagy. In humans, Parkin ubiquitinates mitochondrial substrates
to induce mitophagy. In this study, the ubiquitin isopeptidase Doa4
is shown to be required for mitophagy, indicating for the first
time a mitophagy-related role of ubiquitin in yeast.
Another selective variant of autophagy is PMN, the piecemeal
microautophagy of the nucleus. A specific cargo-recognition in PMN
is indispensable to preserve the integrity of the nucleus.
Therefore, the still unknown nuclear cargo was a focus of interest.
The results of this study point to a specific cargo-recognition of
nucleolar proteins in PMN. Strains expressing different nucleolar
GFP fusion proteins were investigated, some showed PMN-like
structures in microscopy. Furthermore, nucleus-vacuole junctions,
the contact sites between the nucleus and the vacuolar membrane,
are shown to localise near the nucleolus. | de |