In Pichia pastoris, most of the Glycosylphosphatidylinositol (GPI)-anchored proteins are of unknown function. Gcw13, one of these GPI-anchored proteins, was found to exert an inhibitory effect on the growth of the histidine auxotrophic P. pastoris strain GS115 on methanol as the sole carbon source. To investigate the biological function of Gcw13, RNA sequencing (RNA-Seq) was performed to compare the difference of gene expression between GS115 and GCW13-deletion strain D13. RNA-Seq analysis showed that, in strain D13, the expression of genes involved in the methanol utilization pathway or peroxisome biogenesis was not changed, and a high proportion of genes involved in the biosynthesis of amino acids were down-regulated, whereas GAP1, which encodes a general amino acid permease, was significantly up-regulated. Besides, the intracellular concentrations of various amino acids were significantly higher in D13 than that in GS115. We also observed that deletion of GCW13 resulted in more Gapl presented on the cell surface and more active uptake of the toxic proline analogue L-azetidine-2-carboxylate acid (AzC). These results suggest that Gcw13 suppresses the expression of GAP1 and facilitates the endocytosis of Gap1 on methanol, resulting in decreasing Gap1-dependent uptake of amino acids in P. pastoris, which might contribute to the poor growth of GS115 on methanol, (C) 2018 Elsevier Inc. All rights reserved.