Staff Members of the Institute of Biochemistry, TU - Institut für ...

Lot6p – a redox regulated switch of the proteasome
During our previous studies of bacterial quinone reductases, we have also investigated the
biochemical properties of the yeast homolog Lot6p. Despite the availability of a three-dimensional
structure for Lot6p (1T0I), the physiological role of the enzyme was unclear.
Our recent studies have now demonstrated that the enzyme rapidly reduces quinones at the
expense of a reduced nicotinamide cofactor, either NADH or NADPH. In order to further
characterize the cellular role of Lot6p, we have carried out pull-down assays and identified the 20S
core particle of the yeast proteasome as interaction partner. Further studies revealed that this
complex recruits Yap4p, a member of the b-Zip transcription factor family, but only when the
flavin-cofactor of Lot6p is in its reduced state. Oxidation of the flavin leads to dissociation of the
transcription factor and relocalization to the nucleus (see scheme below).
reduced
Lot6p
α
β
β
α
A similar system is known from mammalian cells, where a homologous quinone reductase
(NQO1) binds to the 20S proteasome and recruits important tumor suppressor proteins such as p53
and p73α. Hence, the discovery of a homologous protein interaction in yeast provides an
interesting model system to investigate the molecular basis for protein complex formation and
regulation of proteasomal degradation of transcription factors (postdoctoral project of Ines
Waldner-Scott; thesis project of Sonja Sollner and Venugopal Gudipati).
Zn-dependent Alkylsulfatases
Yap4p
Oxidation by
e.g.
quinones
Hydrolysis of alkylsulfates is an important pathway for soil and other bacteria to mobilize sulfur.
Three classes of sulfatases have been discovered, and the recently elucidated structure of SdsA1
from Pseudomonas aeruginosa revealed that the enzyme belongs to the widely occurring family of
Zn-dependent ß-lactamases. A detailed characterization of the enzyme’s stereochemistry by the
group of Prof. Faber at the University of Graz demonstrated that this enzyme cleaves primary
alklysulfates (preferred substrate is dodecylsulfate) by retention. In other words, the hydroxyl
group attacks the sulfur atom in the course of the reaction (see reaction scheme below). Only
recently a novel enzyme activity could be identified in another Pseudomonas strain, which leads to
hydrolytic cleavage of alkylsulfates via stereoinversion, i. e. the hydroxyl group attacks the carbon
rather than the sulfur atom of the substrate (see scheme below).
10
oxidized
Lot6p
Very slow with oxygen O2 nucleus
oxygen
β
α
β
α
Yap4p
Yap4p