Functional and transcriptome analyses of Hansenula polymorpha

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    Functional and transcriptome analyses of Hansenula polymorpha Hac1p, a key UPR 4

    transcription factor, reveal a critical function in modulating protein N-glycosylation activity 5

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    Running title: Modulation of protein N-glycosylation by HpHac1p 7

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    9 Hye-Yun Moon1,2, Seon Ah Cheon1, Hyunah Kim1, M. O. Agaphonov3, Ohsuk Kwon4, Doo-10

    Byoung Oh4, Jeong-Yoon Kim2,#, and Hyun Ah Kang1,# 11

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    13 1Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-14 756, Korea 15 2Department of Microbiology and Molecular Biology, College of Bioscience and 16 Biotechnology, Chungnam National University, Daejeon 305-764, South Korea 17 3A.N. Bach Institute of Biochemistry of the Russian Academy of Sciences, Moscow, Russia 18 4Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahakro, 19 Yuseong-gu, Daejeon 305-806, Korea 20

    21 22 23 24 #Corresponding author 25 Mailing address for Hyun Ah Kang: Department of Life Science, College of Natural Science, 26 Chung-Ang University, Seoul 156-756, South Korea. Phone: 82-2-820-5863. Fax: 82-2-825-27 5206. E-mail: hyunkang@cau.ac.kr. 28 Mailing address for Jeong-Yoon Kim: Department of Microbiology and Molecular Biology, 29 College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, 30 South Korea. Phone: 82-42-821 -6419. Fax: 82-42-822-7367. E-mail: jykim@cnu.ac.kr. 31 32 33

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    AEM Accepted Manuscript Posted Online 31 July 2015Appl. Environ. Microbiol. doi:10.1128/AEM.01440-15Copyright 2015, American Society for Microbiology. All Rights Reserved.

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    ABSTRACT 35

    Aggregation of misfolded protein in the endoplasmic reticulum (ER) induces a cellular 36

    protective response to ER stress, the unfolded protein response (UPR), which is mediated by 37

    a bZIP transcription factor, Hac1p/Xbp1. In this study, we identified and studied molecular 38

    functions of a HAC1 homolog (HpHAC1) in the thermotolerant yeast Hansenula polymorpha. 39

    We found that the HpHAC1 mRNA contains a nonconventional intron of 177 bp whose 40

    interaction with the 5-UTR is responsible for translational inhibition of the HpHAC1 mRNA. 41

    The H. polymorpha hac1 null mutant strain (Hphac1) grew slowly, even under normal 42

    growth conditions, and were less thermotolerant than the wild-type (WT) strain. The mutant 43

    strain also was more sensitive to cell wall perturbing agents and to UPR-inducing 44

    dithiothreitol (DTT) and tunicamycin (TM). Using comparative transcriptome analysis of the 45

    WT and Hphac1 strains treated with DTT and TM, we identified HpHAC1-dependent core 46

    UPR targets, which included genes involved in protein secretion and processing, particularly 47

    those required for N-linked protein glycosylation. Notably, different glycosylation and 48

    processing patterns of the vacuolar glycoprotein carboxypeptidase Y were observed in the 49

    WT and Hphac1 strains. Moreover, overexpression of active HpHac1p significantly 50

    increased N-linked glycosylation efficiency and TM resistance. Collectively, our results 51

    suggest that the function of HpHac1p is important, not only just for UPR induction, but also 52

    for efficient glycosylation in H. polymorpha. 53

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    INTRODUCTION 56

    The endoplasmic reticulum (ER) represents the central organelle of the eukaryotic cell, in 57

    which the crucial steps of protein folding, modification, and selection for transport to other 58

    compartments take place. Perturbation of ER homeostasis through, for example, the 59

    accumulation of misfolded proteins or defects in the ER membrane, leads to activation of 60

    intracellular signaling pathways referred to as the unfolded protein response (UPR), ER-61

    associated protein degradation (ERAD), and the calnexin cycle (CNX) for glycoprotein 62

    quality control (1). UPR induces multiple protective cellular events required for proper 63

    protein folding and misfolded protein degradation. The underlying regulatory mechanism and 64

    modes of action of UPR has been intensively studied in the traditional yeast Saccharomyces 65

    cerevisiae (2). UPR is mediated by a master transcription factor, Hac1p, in S. cerevisiae (3), 66

    and the synthesis of functionally active Hac1p is triggered via non-conventional splicing of 67

    the HAC1 mRNA, which is mediated by the endonuclease activity of the ER stress sensor, 68

    Ire1p (4). This unusual splicing of HAC1 mRNA induces translation of HAC1 mRNA by 69

    eliminating base-pairing between the 5-untranslated region (5-UTR) and the splicing site (5). 70

    The newly synthesized Hac1p then is shuttled into the nucleus and functions as an active 71

    bZIP transcription factor during ER stress. Hac1p binds to a UPR element (UPRE) within the 72

    promoter regions of its target genes. The consensus motif of UPRE-1, CANCNTG, is found 73

    in the promoters of the KAR2, PDI1, and FKB2 genes in S. cerevisiae (6). 74

    The protein secretion pathways of yeast and filamentous fungi are of special interest to 75

    researchers that are developing industrial protein producers. Understanding the underlying 76

    mechanisms of UPR would provide solid knowledge on the regulation of cellular responses 77

    to protein secretion stress (7). In this aspect, the HAC1 homologs of non-Saccharomyces 78

    yeasts and filamentous fungi that are potential hosts for industrial production of recombinant 79

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    proteins have been identified and characterized. Unlike the S. cerevisiae HAC1 mRNA, 80

    which contains an intron of 252 nt, the hac1/hacA genes encoding the key UPR transcription 81

    factors from the filamentous fungi Trichoderma reesei and Aspergillus nidulans contain 82

    introns that are 20 nt long (8) . Similarly, the Yarrowia lipolytica HAC1 mRNA harbors a 83

    short intron of 29 nt (9). In contrast, the intron of the Pichia pastoris HAC1 mRNA is 322 nt, 84

    which is longer than the S. cerevisieae HAC1 mRNA intron (10). Despite the differences in 85

    intron length, the HAC1 mRNAs of these yeast and filamentous fungi undergo similar, 86

    unconventional splicing reactions to produce functional Hac1 proteins (8, 9, 11). 87

    Hansenula polymorpha is a thermotolerant methylotrophic yeast, which can grow at 88

    temperatures up to 48oC and utilize methanol as a sole carbon and energy source. H. 89

    polymorpha has been a favorable model to study the mechanisms of genetic control of 90

    methanol metabolism and peroxisome biogenesis. In recent decades, H. polymorpha has also 91

    been used as a host for heterologous protein production (12). Particularly, glyco-engineered H. 92

    polymorpha strains have been developed to produce glycoproteins with human-compatible N-93

    glycans based on information on the host-specific structure and biosynthesis pathway of N-94

    linked glycosylation in H. polymorpha (13-15). Likewise, a recent study on the identification 95

    and functional analysis of protein O-mannosyltransferases (PMTs) in H. polymorpha 96

    provided some unique features of PMT proteins in H. polymorpha (16). In this study, to 97

    obtain further details on the gene regulatory networks required for protein secretion and 98

    modification in H. polymorpha, we identified and characterized the HAC1 homolog of H. 99

    polymorpha via functional analysis and genome-wide gene expression profiling. 100

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    MATERIALS AND METHODS 102

    Strains, plasmids, and culture conditions. H. polymorpha and S. cerevisiae strains used in 103

    this study are described in Table S1. Plasmids used in this study are listed in Table S2, and the 104

    primers used for the construction of strains and plasmids are listed in Table 1. Yeast cells 105

    were routinely grown in YPD medium (1% bacto-yeast extract, 2% bacto-peptone, 2% 106

    glucose) or synthetic complete (SC) medium (0.67% yeast nitrogen base without amino acids, 107

    2% glucose, drop-out amino acid mixture supplemented with all required amino acids). 108

    Hphac1 and Hpire1 deletion mutants were constructed in the H. polymorpha DL1 109

    background by fusion PCR and in vivo DNA recombination (15), using the primer sets listed 110

    in Table 1. PCR fragments were introduced into yeast cells, and the transformants carrying 111

    the gene deletion as a result of in vivo DNA recombination were selected first by growth in 112

    minimal media lacking leucine (SC-LEU) and then by PCR screening. 113

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    Construction of HpHAC1 expression vectors. The vectors pDUM2-HA-HAC1s and 115

    pDUM2-HA-HAC1u for expression of HpHac1p tagged at its N-terminus by the epitope 116

    corresponding to amino acids 98-106 of human influenza hemagglutinin (HA), under the 117

    control of the MOX1 promoter, were constructed as follows. The DNA fragment containing a 118

    spliced form of HpHAC1 (HpHAC1s) was amplified from the cDNA of DTT-treated WT H. 119

    polymorpha cells with primers HpHAC1_HA_1F

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