Lactate dehydrogenase is C-terminally extended by stop codon read-through which targets this isoform into the peroxisomes

Abstract

In this work, the functional significance of stop codon read-through was investigated in relation to the human peroxisomes. When the ribosomes translating a messenger RNA encounter a stop codon, they usually stop the translation leading to the release of the polypeptide chain. However, when the translation continues uninterrupted by the erroneous incorporation of an amino acid at the stop codon, it leads to stop codon read-through. Although, this process appears to generate protein variants in viruses, yeasts and fungi, it had not been clear if in human’s read-through proteins have other functions than their parent proteins. Understanding the molecular mechanisms of read-through can be pivotal to treat rare genetic diseases caused due to nonsense mutations. Therefore, in our study we have developed and analysed a computational model which estimates the read-through propensity (RTP) of stop codon contexts (defined as the stop codon and approximately 12 nucleotides in its vicinity). Coupling of this model with another algorithm which predicts proteins targeted to the peroxisomes identified lactate dehydrogenase B (LDHB) variant with a high propensity for read-through and peroxisome localisation. Developing and employing reporter assays and immunofluorescence studies, we have confirmed the generation of a read-through variant called LDHBx which has a functional peroxisome targeting signal (PTS1). Mass spectrometric analysis of LDHB immunoprecipitates from rat tissues identified glyceraldehyde -3-phosphate dehydrogenase (GAPDH) as an interaction partner. Preliminary studies showed piggy-back import of GAPDH inside peroxisomes in the presence of read-through extended LDHBx. We therefore conclude, that the stop codon context of LDHB trigger efficient read-through to generate protein variant with peroxisome targeting. This variant aids in co-import of non-peroxisomal proteins such as GAPDH inside the organelle which we speculate may be involved with LDH in maintenance of redox homeostasis.