Submitted on August 11, 2006
Accepted on July 23, 2007
TWO ISOFORMS OF PSAP/MTCH1 SHARE TWO PROAPOPTOTIC DOMAINS AND MULTIPLE INTERNAL SIGNALS FOR IMPORT INTO THE MITOCHONDRIAL OUTER MEMBRANE
Violeta Lamarca1, Antonio Sanz-Clemente2, Rosaura Maria Perez-Pe3, Maria Jose Martinez-Lorenzo4, Nabil Halaihel5, Pedro Muniesa6, and Jose Alberto Carrodeguas1*
1 Institute for Biocomputation and Physics of Complex Systems, University of Zaragoza, Zaragoza, Zaragoza, Spain
2 Receptor Biology Unit, NINDS Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, United States
3 Biochemistry and Molecular and Cellular Biology, University of Zaragoza, Zaragoza, Zaragoza, Spain
4 Immunology Service, University Clinical Hospital Lozano Blesa, Zaragoza, Zaragoza, Spain
5 Department of Animal Pathology, University of Zaragoza, Zaragoza, Zaragoza, Spain
6 Department of Anatomy, Embriology and Genetics, University of Zaragoza, Zaragoza, Zaragoza, Spain
* To whom correspondence should be addressed. E-mail: carrode{at}unizar.es.
Presenilin 1-associated protein, PSAP, was first identified as a protein that interacts with presenilin 1. It was later reported that PSAP is a mitochondrial protein that induces apoptosis when overexpressed in cultured cells. PSAP is also known as mitochondrial carrier homolog 1, Mtch1. Here we show that there are two proapoptotic PSAP isoforms, generated by alternative splicing, that differ in the length of a hydrophilic loop located between two predicted transmembrane domains. Using RT-PCR and western blot assays we determined that both isoforms are expressed in human and rat tissues as well as in culture cells. Our results indicate that PSAP is an integral mitochondrial outer membrane protein, although it contains a mitochondrial carrier domain conserved in several inner membrane carriers, which partially overlaps one of the predicted transmembrane segments. Deletion of this transmembrane segment impairs mitochondrial import of PSAP. Replacement of this segment with each of two transmembrane domains, with opposite membrane orientations, from an unrelated protein indicated that one of them allowed mitochondrial localization of the PSAP mutant whereas the other one did not. Our interpretation of these results is that PSAP contains multiple mitochondrial targeting motifs dispersed along the protein but that a transmembrane domain in the correct position and orientation is necessary for membrane insertion. The amino acid sequence within this transmembrane domain may also be important. Furthermore, two independent regions in the amino terminal side of the protein are responsible for its proapoptotic activity. Possible implications of these findings in PSAP function are discussed.
