Hansenula polymorpha Hac1p Is Critical to Protein N-Glycosylation Activity Modulation, as Revealed by Functional and Transcriptomic Analyses

Abstract

Aggregation of misfolded protein in the endoplasmic reticulum (ER) induces a cellular protective response to ER stress, the unfolded protein response (UPR), which is mediated by a basic leucine zipper (bZIP) transcription factor, Hac1p/Xbp1. In this study, we identified and studied the molecular functions of a HAC1 homolog from the thermotolerant yeast Hansenula polymorpha (HpHAC1). We found that the HpHAC1 mRNA contains a nonconventional intron of 177 bp whose interaction with the 5' untranslated region is responsible for the translational inhibition of the HpHAC1 mRNA. The H. polymorpha hac1-null (Hphac1Δ) mutant strain grew slowly, even under normal growth conditions, and was less thermotolerant than the wild-type (WT) strain. The mutant strain was also more sensitive to cell wall-perturbing agents and to the UPR-inducing agents dithiothreitol (DTT) and tunicamycin (TM). Using comparative transcriptome analysis of the WT and Hphac1Δ strains treated with DTT and TM, we identified HpHAC1-dependent core UPR targets, which included genes involved in protein secretion and processing, particularly those required for N-linked protein glycosylation. Notably, different glycosylation and processing patterns of the vacuolar glycoprotein carboxypeptidase Y were observed in the WT and Hphac1Δ strains. Moreover, overexpression of active HpHac1p significantly increased the N-linked glycosylation efficiency and TM resistance. Collectively, our results suggest that the function of HpHac1p is important not only for UPR induction but also for efficient glycosylation in H. polymorpha.

FIG 1

Analysis of Ire1p-mediated H. polymorpha HAC1 splicing under UPR-induced conditions. (A) RT-PCR analysis of HpHAC1 splicing upon UPR induction by TM treatment in H. polymorpha DL1-L WT and Hpire1Δ mutant cells. (B) Diagram showing the binding site of the primers used for RT-PCR analysis. HpHAC1^u, unspliced form of the HpHAC1 transcript; HpHAC1^s, spliced form of the HpHAC1 transcript. (C) Structural organization of HpHac1p and ScHac1p translated after unconventional splicing of HAC1 mRNAs. Dark vertical lines, PEST domains; small grid lines, transcriptional activation domain (TAD); aa, amino acids.

FIG 2

Analysis of HpHac1p expression in the H. polymorpha WT and ire1-disrupted strains. (A) Predicted secondary structure of the intron in the HpHAC1 mRNA loop and putative sites of cleavage by Ire1p. The HpHAC1 5′ UTR and intron can interact by base paring. (B, C) Western blot analysis of the N-terminally HA-tagged HpHac1^u and HpHac1^s proteins under the control of the native promoter (B) or under the control of the MOX promoter (C). The H. polymorpha cells were cultivated in YPD or YPM (1% Bacto yeast extract, 2% Bacto peptone, 2% methanol) at 37°C for 8 h, and 10 μg of total cell extracts was analyzed by immunoblotting with anti-HA antibody.

FIG 3

Growth phenotype analyses of H. polymorpha hac1Δ strains. The results of thermotolerance analysis (A), inositol auxotrophy analysis (B), and analysis of sensitivity to cell wall perturbation (C, D) of the H. polymorpha WT [Hp(WT)] and Hphac1Δ strains are shown. The S. cerevisiae WT [Sc(WT)] and hac1Δ strains were spotted as controls. Yeast cells were grown to stationary phase in YPD medium, and a 10-fold dilution series was spotted onto a YPD plate at 30°C, 37°C, and 45°C or onto YPD plates containing 10 mM caffeine, 0.01% SDS, or 45 μg/ml CFW without or with the addition of 0.5 M KCl. Ino, inositol. (E) Effect of HpHAC1^s and HpHAC1^sΔPEST (HpHAC1^s-ΔP) expression on ER stress resistance.

FIG 4

Transcriptome profile of the Hphac1 disruption mutant. (A) Growth of WT and Hphac1Δ mutant cells in YPD medium at 37°C. (B) Scatter plots reflecting the differential expression profiles of the WT and Hphac1Δ strains cultivated in YPD under normal condition. Total RNAs were obtained from WT strain DL1 and Hphac1Δ mutant cells grown to log phase (OD₆₀₀, ∼0.5) in YPD medium and subjected to microarray analysis using a whole-genome microarray. Red spots, genes up- or downregulated more than 2-fold in the Hphac1 mutant compared to their expression in the WT strain; green spots, genes showing less than 2-fold differential expression in the WT and mutant strains. (C) Genes up- and downregulated in the Hphac1Δ mutant compared to their expression in the WT under normal growth conditions on YPD. The annotated genes were functionally categorized according to the Munich Information Centre for Protein Sequences (MIPS). Pa, Pichia angusta; Ca, Candida albicans; Kl, Kluyveromyces lactis.

FIG 5

Identification of HpHAC1-dependent core UPR genes. (A) Schematic diagram of sample preparation for microarray analysis. (B) Venn diagrams illustrating the overlapped genes up- and downregulated in the WT and Hphac1Δ strains under conditions with TM and DTT treatment (left) and the functional categories of the core UPR target genes based on the Munich Information Centre for Protein Sequences (right). 1-1, amino acid metabolism; 1-2, nitrogen sulfur and selenium metabolism; 1-3, nucleotide/nucleoside/nucleobase metabolism; 1-4, phosphate metabolism; 1-5, C compound and carbohydrate metabolism; 2, energy; 3, cell cycle and DNA processing; 4, transcription; 6, protein fate; 7, protein with binding function or cofactor requirement; 9, cellular transport, transport facilitation, and transport routes; 14, unclassified proteins.

FIG 6

Quantitative real-time PCR analyses of HpHAC1-dependent core UPR genes. H. polymorpha DL1-L (WT) and Hphac1Δ mutant cells grown to log phase (OD₆₀₀, ∼0.3) in YPD medium were transferred to YPD supplemented with 5 mM DTT or 5 μg/ml TM. RNA samples from the WT treated with TM (squares) or DTT (diamonds) and from the Hphac1Δ mutant treated with TM (triangles) or DTT (×) were prepared at the times indicated on the x axes (in minutes) and analyzed by qRT-PCR. The relative levels of induced expression are expressed as the ratio of the mRNA level of each gene to the mRNA level of HpACT1.

FIG 7

Comparative analysis of CPY protein processing in the WT, Hphac1Δ mutant, and HpHAC1^s-overexpressing strains. (A) Time course analysis of HpCPY processing after TM treatment. The H. polymorpha WT and Hphac1Δ mutant were grown in YPD to exponential phase (OD₆₀₀ ∼0.5) before being treated with 5 μg/ml TM for 3 h. Western blot analysis was performed with total intracellular protein extracts using anti-HpCPY antibody. (B) Effect of HpHAC1^s on N-glycosylation of ScCPY proteins. H. polymorpha DL1-LdU strains harboring the ScCPY expression vector pDLGAP-ScCPY with or without the HA-HpHAC1^s expression vector pDUM2-HA-HAC1^s were incubated in YPM medium containing 0.5% methanol for 12 h. Total cell extracts were analyzed by immunoblotting with anti-ScCPY antibody.