SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

1999
P7BP1 E3 ubiquitin ligase controls the quality of PTS2 receptor, Pex7p.
Y. Miyauchi 1 , Y. Fujiki 1 ; 1 Kyushu University, Fukuoka, Japan
SUNDAY
Peroxisomes are ubiquitous single membrane-bound organelles that contain about 100 different
enzymes catalyzing various metabolic pathways such as fatty-acid ƒÀ-oxidation and
etherglycerolipid synthesis. Peroxisomes are formed by growth and division of preexisting
peroxisomes after posttranslational import of newly synthesized peroxisomal matrix and
membrane proteins. Two distinct signals, peroxisomal targeting signal type 1 (PTS1) and PTS2,
direct proteins to the peroxisomal matrix. PEX5 and PEX7 encode the cytosolic receptors for
PTS1 and PTS2, respectively. Mono-Ubiquitination of Pex5p is required for its export from
peroxisomal membrane to the cytosol in yeast to mammals. However, regulation of the function
and transport of Pex7p remains undefined.
We first investigated whether or not Pex7p is ubiquitinated and degraded by proteasomes in
cells. Addition of a proteasome inhibitor, MG132 to cell culture delayed the turnover rate of
Pex7p. We also attempted to isolate any potential Pex7p-binding proteins potentially involved in
the degradation pathway of Pex7p. From a cell line stably expressing FLAG-Pex7p, we isolated
several Pex7p-binding proteins, termed P7BPs: Pex7p binding proteins. Suppression of P7BP1
by RNAi resulted in a delay of Pex7p turnover rate, as observed in the MG132-treated cells,
hence implying that P7BP1 plays a role in Pex7p ubiquitination. Furthermore, we also show that
the degradation of dysfunctional Pex7p mediated by P7BP1 is crucially required for the
maintenance of normal PTS2 import. Our findings may define a mechanism underlying Pex7p
degradation and its importance in regulating PTS2 import.
2000
A System to Quantify the Differential Import of Peroxisomal Matrix Protein by Measuring
Fluorescence Intensity.
M. Noguchi 1 , Y. Fujiki 1,2 ; 1 Graduate School of Systems Life Sciences, Kyushu University,
Fukuoka, Japan, 2 CREST, JST
Fourteen distinct peroxins are identified as essential factors for peroxisome biogenesis in
mammals, of which ten are involved in peroxisomal import of matrix proteins. Peroxisomal
matrix protein import is regulated by various cellular factors. However, mechanisms underlying
such regulations are poorly understood, partly, if not all, due to the lack of quantitative detection
method with high resolution. Here, we developed a monitoring system that utilizes the
differences in the stability of fluorescent reporters localized between peroxisomes and the
cytosol. An FKBP12 variant, termed destruction domain (DD), is rapidly and constitutively
degraded by proteasomes when expressed in mammalian cells. Degradation of DD is reversibly
protected by addition of a specific synthetic ligand. In the absence of the ligand, a reporter DD-
EGFP-PTS1, EGFP fused with the DD and peroxisomal targeting signal 1, is largely degraded
in the cytosol prior to entering peroxisomes in wild-type cells. In contrast, in the presence of the
ligand the reporter becomes stable and translocates into peroxisomes. Upon withdrawal of the
ligand, the reporter in peroxisomes remains intact, whilst that in the cytosol is rapidly degraded.
Thus, peroxisomal protein import can be readily quantified by simply measuring the
fluorescence intensity of whole cells.