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

Abstract

dAggregation 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 (Hphac1⌬) 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 Hphac1⌬ 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 Hphac1⌬ 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. The endoplasmic reticulum (ER) represents the central organelle of the eukaryotic cell in which the crucial steps of protein folding, modification, and selection for transport to other compartments take place. Perturbation of ER homeostasis through, for example, the accumulation of misfolded proteins or defects in the ER membrane leads to the activation of intracellular signaling pathways referred to as the unfolded protein response (UPR), ERassociated protein degradation (ERAD), and the calnexin cycle (CNX) for glycoprotein quality control (1). UPR induces multiple protective cellular events required for proper protein folding and misfolded protein degradation. The underlying regulatory mechanism and modes of action of UPR have been intensively studied in the traditional yeast Saccharomyces cerevisiae (2). UPR is mediated by a master transcription factor, Hac1p, in S. cerevisiae (ScHac1p) (3), and the synthesis of functionally active Hac1p is triggered via nonconventional splicing of the HAC1 mRNA, which is mediated by the endonuclease activity of the ER stress sensor, Ire1p (4). This unusual splicing of HAC1 mRNA induces the translation of HAC1 mRNA by eliminating base pairing between the 5= untranslated region (UTR) and the splicing site (5). The newly synthesized Hac1p is then shuttled into the nucleus and functions as an active basic leucine zipper (bZIP) transcription factor during ER stress. Hac1p binds to a UPR element (UPRE) within the pr...

Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins

“…Prolonged activation of the UPR can result in so-called ER-phagy, when parts of the ER are removed to relieve the ER stress and remove the misfolded proteins (Kruse et al 2006 ). In addition, a sustained activation of UPR can impair cellular growth, as reported for different yeasts (Cox et al 1993 ; Kawahara et al 1997 ; Chawla et al 2011 ; Cheon et al 2011 ; Miyazaki et al 2013 ; Moon et al 2015 ). In P. pastoris , slower growth was observed in the case of co-expression of HAC1 in strains producing xylanase A from Bacillus halodurans or human lysozyme (Lin et al 2013 ; Liu et al 2020 ), of PDI1 in a strain producing α-glucosidase from Aspergillus niger (Liu et al 2013 ), or of ERO1 in a strain producing Rhizomucor miehei lipase (Huang et al 2020 ).…”

“…In P. pastoris , increased expression or secretion of many different recombinant proteins resulted from co-expression of the following: the ER-chaperone Kar2p or protein disulfide isomerase Pdi1 (Inan et al 2006 ; Damasceno et al 2007 ; Sallada et al 2019 ), enzymes involved in the ER redox control and oxidative stress such as Ero1, Gpx1, Aha1, or Ypt6 (Sha et al 2013c ; Ben Azoun et al 2016a ; Huangfu et al 2016 ; Sallada et al 2019 ), the UPR transcription factor Hac1p (Guerfal et al 2010 ; Vogl et al 2014 ; Li et al 2015 ; Krainer et al 2016 ; Huang et al 2017 ; Han et al 2020 ; Liu et al 2020 ), the kinase/RNase Ire1p (Yu et al 2020 ), or new co-chaperones (Huangfu et al 2016 ) (Table 2 ). Glycosylation activity was also increased (Moon et al 2015 ) or product homogeneity and processing of the secretion α-factor were improved (Guerfal et al 2010 ). Recently, three novel folding factors, Mpd1p (member of the PDI family), Pdi2p (protein disulfide isomerase), and Sil1p (nucleotide exchange factor for the ER lumenal Hsp70 chaperone Kar2p), were characterized and their genes co-expressed in P. pastoris (Duan et al 2019 ).…”

Identification of an Exceptionally Long Intron in the HAC1 Gene of Candida parapsilosis

“…2B ). Orthologs of several of these genes are also hac1 -dependent in other species such as in C. albicans (6 genes [ 7 ]), S. cerevisiae (7 genes [ 1 , 28 , 29 ]) and H. polymorpha (4 genes [ 30 ]). Three are required for virulence in C. albicans : PMT1 ( CPAR2_704010 ), PMT4 ( CPAR2_104900 ) and SERP1 ( CPAR2_102440 ) ( 31 , 32 ).…”

Yeasts for Global Happiness: report of the 14th International Congress on Yeasts (ICY14) held in Awaji Island

“…; Moon et al . ) (day 5); (x) ‘Metabolic engineering of Saccharomyces cerevisiae for 1st generation ethanol and anaerobic glycerol production’ by Dr. Andriy Sibirny (NAS of Ukraine, Ukraine; Fig. M) regarding optimization of the productivity of ethanol or glycerol from glucose via specific gene modifications (Semkiv et al .…”