Enhanced folding capacity of green fluorescent protein facilitates vacuolar trafficking of carboxypeptidase Y.

Efficient transport of secretory proteins through the endoplasmic reticulum (ER) is essential for their correct localization and cellular function. Misfolded proteins are retained in the ER and eliminated via ER-associated degradation (ERAD), but whether enhanced folding efficiency promotes ER exit and subsequent trafficking remains unclear. To address this question,Arabidopsis carboxypeptidase Y (CPY), a vacuolar protein that passes through the ER, Golgi apparatus, and prevacuolar compartment en route to the vacuole, was used. CPY was fused to three green fluorescent protein (GFP) variants differing in folding properties: standard GFP, superfolder GFP (sfGFP) with enhanced folding capacity, and aggregation-prone GFP[V30A]. As expected, CPY:GFP[V30A] failed to reach the vacuole and accumulated mainly as the precursor form. In contrast, CPY:sfGFP displayed markedly improved vacuolar trafficking compared with CPY:GFP, indicating that enhanced folding efficiency facilitates ER exit and downstream transport. Similar results with CPY95, a truncated CPY variant, further suggest that this effect is independent of protein length. These findings uncover a previously unrecognized role for folding efficiency in regulating secretory protein trafficking, demonstrating that protein folding status can directly influence the efficiency of ER export and vacuolar delivery.