Abstract
Purpose:
The purpose of this study was to determine whether the signal peptide is alone responsible for the intracellular mislocalization of the age-related macular degeneration (AMD)-related variant Cystatin C in the retinal pigmented epithelium (RPE). Variant B cystatin C is generated by a point mutation in its leader sequence. As a consequence, this protein has been reported to divert from the secretory pathway to an unusual mitochondria association.
Methods:
Constructs encoding green fluorescent protein (eGFP) N-terminally linked to the leader sequence from wild-type and variant B cystatin C were made. RPE cell lines expressing these constructs were used to analyze the intracellular distribution of the eGFP by cell imaging and immunoblotting. Cell fractions obtained by differential centrifugation and mitochondrial fractions obtained by magnetic separation were used in the analysis. eGFP secretion in conditioned media was analyzed by Western blotting to further define the role of the leader sequence in targeting the protein to the secretory pathway.
Results:
Both wild-type and variant leader sequences directed the eGFP to the ER/secretory pathway in stark contrast to the leaderless eGFP. No significant difference in protein distribution between the two signal peptides was observed and in particular variant B leader peptide did not direct eGFP to mitochondria. However, although both constructs targeted eGFP for secretion, a reduction in the efficiency of this process was observed in the case of variant signal peptide. The experimental data supports the hypothesis that the reduced secretion of the variant protein is due to unfavourable interaction of its leader sequence (less hydrophobic) with components of the ER translocation channel and not to its loss of ER targeting.
Conclusions:
The variant signal peptide is therefore not responsible alone for the mitochondria-association of variant B cystatin C. We conclude that this mislocalization is most likely due to changes of the chemical-physical properties of the whole precursor cystatin C protein sequence that probably alters the folding/conformation of cystatin C and, conversely, the conformational change may determine interactions with proteins involved in mitochondria localization. Understanding the molecular mechanism underlying this mistrafficking will help understand how it contributes to the impairment of RPE function.
Keywords: 412 age-related macular degeneration •
701 retinal pigment epithelium •
600 mitochondria