Affiliation:
1. Molecular Biology Program, Sloan Kettering Institute , New York, New York, USA
2. Gerstner Sloan Kettering Graduate School of Biomedical Sciences , New York, New York, USA
3. Department of Microbiology and Immunology, Weill Cornell Medical College , New York, New York, USA
4. Institute of Organic Chemistry and Centre for Integrative Biological Signaling Studies, University of Freiburg , Freiburg, Germany
5. Spemann Graduate School of Biology and Medicine, University of Freiburg , Freiburg, Germany
Abstract
ABSTRACT
Inositol pyrophosphate 1,5-IP
8
is a signaling molecule that regulates phosphate and polyphosphate homeostasis in the fission yeast
Schizosaccharomyces pombe
. 1,5-IP
8
levels are dictated by a balance between the Asp1 kinase domain that converts 5-IP
7
to 1,5-IP
8
and two pyrophosphatases—the Asp1 pyrophosphatase domain (histidine acid phosphatase family) and the Aps1 pyrophosphatase enzyme (Nudix family)—that hydrolyze the β-phosphates of 1,5-IP
8
. Here, we characterize
S. pombe
Siw14 (SpSiw14), a cysteinyl-phosphatase family member and a homolog of
Saccharomyces cerevisiae
Siw14, as a third fission yeast pyrophosphatase implicated in inositol pyrophosphate catabolism. We find that SpSiw14’s substrate repertoire embraces inorganic pyrophosphate, inorganic polyphosphate, and the inositol pyrophosphates 5-IP
7
, 1-IP
7
, and 1,5-IP
8
, in addition to the generic substrate
p
-nitrophenylphosphate. Genetic analyses revealed that (i) elimination of the SpSiw14 protein or inactivation of the SpSiw14 pyrophosphatase by the C189S mutation had no effect on
S. pombe
growth but was lethal in the absence of Aps1 and (ii) the synthetic lethality of
siw14
∆
aps1
∆ depended on the synthesis of 1,5-IP
8
by the Asp1 kinase. We conclude that SpSiw14 and Aps1 pyrophosphatases have essential but redundant functions in fission yeast, and that their synthetic lethality is a consequence of the toxic effects of too much 1,5-IP
8
. Suppression of
siw14
∆
aps1
∆ lethality by loss-of-function mutations of components of the fission yeast 3′-processing/termination machinery fortifies the case for overzealous transcription termination as the basis for 1,5-IP
8
toxicosis.
IMPORTANCE
The inositol pyrophosphate signaling molecule 1,5-IP
8
modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3′-processing and transcription termination. Cellular 1,5-IP
8
levels are determined by a balance between the activities of the inositol polyphosphate kinase Asp1 and several inositol pyrophosphatase enzymes. Here, we characterize
Schizosaccharomyces pombe
Siw14 (SpSiw14) as a cysteinyl-phosphatase-family pyrophosphatase enzyme capable of hydrolyzing the phosphoanhydride substrates inorganic pyrophosphate, inorganic polyphosphate, and inositol pyrophosphates 5-IP
7
, 1-IP
7
, and 1,5-IP
8
. Genetic analyses implicate SpSiw14 in 1,5-IP
8
catabolism
in vivo
, insofar as: loss of SpSiw14 activity is lethal in the absence of the Nudix-type inositol pyrophosphatase enzyme Aps1; and
siw14
∆
aps1
∆ lethality depends on synthesis of 1,5-IP
8
by the Asp1 kinase. Suppression of
siw14
∆
aps1
∆ lethality by loss-of-function mutations of 3′-processing/termination factors points to precocious transcription termination as the cause of 1,5-IP
8
toxicosis.
Funder
HHS | NIH | National Institute of General Medical Sciences
National Science Foundation
Deutsche Forschungsgemeinschaft
Publisher
American Society for Microbiology