Molecular cloning and characterization of a Pichia pastoris ortholog of the yeast Golgi GDP-mannose transporter gene

Abstract

There are two structural profiles in the yeast Golgi. The Golgi of Saccharomyces cerevisiae is composed of a number of vesicular compartments dispersed in the cytoplasm as recognized by a large number of Golgi marker proteins. In contrast, the Golgi of Pichia pastoris was reported to be organized in a small number of stacked cisternae located near the transitional endoplasmic reticulum (tER) sites by electron microscopy and immunofluorescent staining of a few marker proteins. The guanosine diphosphate (GDP)-mannose transporter (GMT) is an essential component in the yeast Golgi apparatus. We isolated an ortholog of the GMT gene of P. pastoris and visualized the gene product by epitope tagging to verify the structural characteristics of the Golgi. The tagged product in P. pastoris cell was observed in rod-like compartments in which Och1 mannosyltransferase was also found and the tER marker Sec12 and Sec13 proteins localized very close to them. The present results add further evidence of the restricted localization of the Golgi in P. pastoris cell.

Functional YFP-tagging of the essential GDP-mannose transporter reveals an important role for the secretion related small GTPase SrgC protein in maintenance of Golgi bodies in Aspergillus niger

“…In this strain, GmtA localization was monitored by fluorescent microscopy revealing the YFP::GmtA protein localized in punctate spots (Fig 5A). Our results are consistent with what has already been illustrated for yeast and filamentous fungi as a typical GDP-mannose transporter, and other Golgi markers, punctate cellular distribution (Dean et al 1997;Gao & Dean 2000;Nishikawa et al 2002b;Abe et al 2004;Arakawa et al 2006;Breakspear et al 2007;Jackson-Hayes et al 2008;Pantazopoulou & Peñalva 2009). The identification of a putative lysine cluster in the C-terminal region (Fig 2, indicated with :) which has been implicated as responsible for location in the Golgi (Abe et al 2004) led us to create a YFP-tagged GmtA construct in which the fluorescent protein located in the N-terminal, in order not to disturb this potential Golgi retention site.…”

Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity

“…Inactivation of related transporters in mammals, yeast and protozoa has been shown to cause problems in morphology or/and virulence (Hirschberg, 1998;Arakawa et al 2006). Cryptococcus neoformans and A. nidulans both have two GDP-mannose transporters, called Gmt1/Gmt2 and GmtA/GmtB, respectively.…”

Cryptococcus neoformans Dual GDP-Mannose Transporters and Their Role in Biology and Virulence

“…Viable cells that express partly functional mutant Vrg4 have been reported, but these strains show defects in both N-and O-linked protein glycosylation and are more sensitive than their wild-type counterparts to cell wall stress (12,13,19). Single VRG4 homologs have been identified in multiple fungi, including Pichia pastoris (20), Aspergillus niger (21), Aspergillus fumigatus (22), Candida glabrata (23), and Candida albicans (24); all of them complement S. cerevisiae vrg4 mutants. While most of these genes, like the S. cerevisiae VRG4 gene, are essential, it is notable that A. fumigatus cells lacking GmtA are viable although significantly impaired (22).…”