Characterization of mouse Clpp protease cDNA, gene, and protein
Brage S. Andresen,
* Thomas J. Corydon,
* Mette Wilsbech,
Lisbeth D. Schroeder,
Tina F. Hindkjær,
Research Unit for Molecular Medicine, Aarhus University Hospital and Faculty of Health Science, Skejby Sygehus, DK 8200 Aarhus N., Denmark
Institute of Human Genetics, Aarhus University, 8000 Aarhus C, Denmark
Received: 25 June 1999 / Accepted: 9 December 1999
Abstract. Mutations that cause accumulation or rapid degradation
owing to protein misfolding are a frequent cause of inherited dis-
ease in humans. In Escherichia coli, Clpp protease is one of the
components of the protein quality control system that handles mis-
folded proteins. In the present study, we have characterized the
mouse Clpp cDNA sequence, the organization of the mouse gene,
the chromosomal localization, and the tissue-specific expression
pattern. Moreover, the cellular localization and processing of
mouse Clpp was studied by overexpression in transfected eukary-
otic cells. Our results indicate that mouse and human Clpp have
similar roles, and they provide the molecular basis for establishing
a Clpp knockout mouse and to study its phenotype, thereby shed-
ding light on a possible role of Clpp in human disease.
It is well known that impaired folding, leading to accumulation or
degradation of misfolded mutant protein, is the major molecular
defect mechanism of a large proportion of mutations that lead to
inherited disease (Taubes 1996; Brooks 1997; Bross et al. 1998,
1999). Despite this, little is known about the proteins involved in
degradation of misfolded proteins in humans, and most of our
knowledge is based on studies performed in yeast and E. coli
(Suzuki et al. 1997). In E. coli, Clpp constitutes part of a protein
quality control system in which chaperones promote protein fold-
ing and proteases degrade misfolded proteins (Gottesman et al.
We have for many years investigated inherited defects in very-
long-chain acyl-CoA dehydrogenase (VLCAD), medium-chain
acyl-CoA dehydrogenase (MCAD) and short-chain acyl-CoA de-
hydrogenase (SCAD). We have shown that most disease-causing
mutations in these mitochondrial enzymes affect the folding and
degradation of the mutant proteins (Andresen et al. 1996, 1997,
1999; Bross et al. 1998; Gregersen et al. 1998; Corydon et al.
1998a). To enable further investigations into the molecular pathol-
ogy of these and other diseases, we are interested in identifying
components of the mammalian mitochondrial protein quality con-
trol system. As an initial approach, we cloned and characterized a
human mitochondrial homolog of the E. coli Clpp protease (Bross
et al. 1995; Corydon et al. 1998b). A powerful approach in this line
of experiments would be to study the phenotype of a Clpp knock-
out mouse and its handling of transgenetically expressed mutant
Recently, another protease, named paraplegin (Casari et al.
1998), was identified in human mitochondria. Inherited defects in
paraplegin lead to hereditary spastic paraplegia in humans. This
illustrates that defects in mitochondrial proteases may lead to dis-
ease by themselves, and it is thus also probable that inherited
defects in Clpp may underlie disease in humans. Studies of a Clpp
knockout mouse may give clues to the disease phenotype of Clpp
deficiency in humans.
In the present study we have characterized the mouse Clpp
cDNA sequence, the organization of the gene, and its chromosom-
al localization. Moreover, we characterized the tissue-specific ex-
pression pattern of mClpp mRNA and investigated the mClpp
protein after overexpression in Chang cells.
Materials and methods
Characterization of mClpp cDNA and genomic sequence.
human Clpp cDNA-specific primers complementary to either the sense or
the anti-sense strand were used in PCR (primer sequences are available on
request: firstname.lastname@example.org). Rapid Amplification of cDNA Ends (RACE)
was performed with the following primers [adaptor-specific primers: AP1
and AP2 (Clontech, Palo Alto, Calif.); mClpp1as: 5Ј-CTCGCGTCG-
CAGTTCCATGCAGGC-3Ј; mClpp3as: 5Ј-CGCTGCGCTGGAAAGTG-
AGC-3Ј; mClpp1s: 5Ј-CCCAATTCCAGAATCATGATCCACCAGC-3Ј;
mClpp3s: 5Ј-GGTTCTGGTCCACCCTCCCCAGG-3Ј]. All PCR’s were
carried out as recommended by the manufacturer (Marathon cDNA am-
plification kit, Clontech) with double-stranded adaptor-ligated cDNA from
adult male BALB/c mouse heart (Clontech) as template. PCR products
were subjected to direct bidirectional cycle sequencing with DNA dye
terminator sequencing kits (TaqFS and BigDye, Perkin-Elmer) in an ABI
Catalyst 800 Molecular Biology LabStation (Applied Biosystems, Foster
City, CA). Sequence reactions were run on semi-automated ABI 373A and
ABI 377 sequencers (Applied Biosystems). Two primers (sense: 5Ј-
GGCTGGGGAGGTACTTC-AGGAGTAAGAACG-3; antisense: 5Ј-CA-
TGGGGCACTAGTGAGGGAGCCACTG-3Ј) located in intron 3 of the
mouse Clpp gene were used to screen a BAC library from 129/Svj mouse
(Genome Systems, St. Louis, Mo. USA). Preparation of BAC DNA was
performed with the KB-Magnum kit (Genome Systems).
Northern and dot blot analysis.
Two nylon filters containing similar
amounts of poly-(A)+ mRNA from different mouse tissues (Clontech)
were used for Northern hybridization, together with a mouse RNA master
harboring normalized loading of poly-(A)+ mRNA from 22 differ-
ent mouse tissues and 7 different control RNA’s and DNA’s (Clontech).
Probes recognizing mouse VLCAD (GeneBank/EMBL accession no.
U07159), and SCAD (GeneBank/EMBL accesssion no. L11163) mRNA’s
were generated by PCR amplification of mouse heart cDNA as fragments
of 2059 bp (VLCAD), 1318 bp (MCAD), and 1219 bp (SCAD) harboring
the entire protein coding regions. The mClpp probe was obtained by cutting
out the entire 936-bp-long mouse Clpp cDNA insert from the pmClpp1A
plasmid (see below) with EcoRI and HindIII. Prior to labeling, the frag-
ments were analyzed by agarose gel electrophoresis, the correctly sized
fragments were cut out of the gel, purified (Qiagen gel extraction kit), and
The nucleotide sequence data reported in this paper have been submitted to
the EMBL Data Bank and have been assigned the accession numbers:
AJ005253, AJ012249–AJ012253, AJ238605, and AJ238606.
* These authors contributed equally to this work.
Correspondence to: B.S. Andresen; email:email@example.com
Mammalian Genome 11, 275–280 (2000).
© Springer-Verlag New York Inc. 2000