The sorting signals for peroxisomal membrane-bound ascorbate peroxidase are within its C-terminal tail

Area of Science:

• Cell Biology
• Molecular Biology
• Protein Trafficking

Background:

• Peroxisomal ascorbate peroxidase (APX) is a key integral membrane protein involved in NAD+ regeneration and H2O2 detoxification.
• APX is sorted post-translationally to peroxisomes via a novel peroxisomal endoplasmic reticulum (pER) network.
• Understanding the molecular signals governing APX sorting is crucial for elucidating peroxisome biogenesis.

Purpose of the Study:

• To identify the molecular signals responsible for the pER-mediated sorting of peroxisomal APX.
• To investigate the role of the C-terminal tail and transmembrane domain (TMD) in APX localization.

Main Methods:

• Site-specific amino acid substitutions and deletions within the C-terminal region of APX.
• Fusion constructs using chloramphenicol acetyltransferase (CAT) to test sorting signals.
• Analysis of protein localization to peroxisomes via the pER pathway.

Main Results:

• Mutations in the C-terminal eight amino acids, including a positively charged domain, abolished APX sorting to peroxisomes.
• The C-terminal tail alone was insufficient for sorting, but when combined with the APX TMD, it directed CAT to peroxisomes via pER.
• Replacement of the APX TMD with an artificial TMD still allowed pER sorting, indicating the TMD itself lacks essential sorting information but provides necessary context.

Conclusions:

• The C-terminal tail of peroxisomal APX contains essential sorting signals for pER-mediated peroxisome targeting.
• The TMD of APX is not a sorting signal itself but is critical for the proper function of the C-terminal sorting signal.
• The positively charged domain within the C-terminal tail acts as an overlapping pER sorting signal and a peroxisome targeting signal type 2.