The Hordeum vulgare cysteine protease HvPAP14 plays a role in degradation of chloroplast proteins.

Chloroplast protein degradation was found to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases were found to be highly expressed during leaf senescence. It remained however unclear where they participate in chloroplast protein degradation. HvPAP14, belonging to the C1A family of cysteine proteases was identified in senescing barley (Hordeumvulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was employed to identify subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed HvPAP14 mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme is activated by low pH being in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins, PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in normal turnover of chloroplast proteins and might have a function for bulk protein degradation during leaf senescence.