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Vol. 10, Issue 5, 1353-1366, May 1999
and
*Department of Anatomy and Cell Biology, University of Florida
College of Medicine, Gainesville, Florida 32610; and
Cytosolic and peroxisomal enzymes necessary for methanol
assimilation are synthesized when Pichia pastoris is
grown in methanol. Upon adaptation from methanol to a glucose
environment, these enzymes are rapidly and selectively sequestered and
degraded within the yeast vacuole. Sequestration begins when the
vacuole changes shape and surrounds the peroxisomes. The opposing
membranes then fuse, engulfing the peroxisome. In this study, we have
characterized a mutant cell line (glucose-induced
selective autophagy),
gsa7, which is defective in glucose-induced selective
autophagy of peroxisomes, and have identified the GSA7
gene. Upon glucose adaptation, gsa7 cells were unable to
degrade peroxisomal alcohol oxidase. We observed that the peroxisomes
were surrounded by the vacuole, but complete uptake into the vacuole
did not occur. Therefore, we propose that GSA7 is not
required for initiation of autophagy but is required for bringing the
opposing vacuolar membranes together for homotypic fusion, thereby
completing peroxisome sequestration. By sequencing the genomic DNA
fragment that complemented the gsa7 phenotype, we have
found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes,
E1. The knockout mutant gsa7Department of Cell Biology, Institute for Cancer
Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo,
Norway
had an identical
phenotype to gsa7, and both mutants were rescued by an
epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a
GSA7 homolog, APG7, a protein required
for autophagy in Saccharomyces cerevisiae, was capable
of rescuing gsa7. We have sequenced the human homolog of
GSA7 and have shown many regions of identity between the
yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and
UBA3). When either of these sites was mutated, the
resulting mutants [Gsa7(ATP)-HA and Gsa7(C518S)-HA] were unable to
rescue gsa7 cells. We provide evidence to suggest that
Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein.
This conjugate was not observed in cells expressing Gsa7(ATP)-HA or
in cells expressing Gsa7(C518S)-HA. Our results suggest that this
unique E1-like enzyme is required for homotypic membrane fusion, a late
event in the sequestration of peroxisomes by the vacuole.
Corresponding author. E-mail address:
dunn{at}anatomy.med.ufl.edu.
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