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The addition of mannose residues to glycoproteins and glycolipids in the Golgi is carried out by mannosyltransferases. Their activity depends on the presence of GDP-mannose in the lumen of the Golgi. The transport of GDP-mannose (mannosyl donor) into the Golgi requires a specific nucleotide sugar transport present in the Golgi membrane. Here, we report the identification and functional characterization of the putative GDP-mannose transporter in Aspergillus niger, encoded by the gmtA gene (An17g02140). The single GDP-mannose transporter was identified in the A. niger genome and deletion analysis showed that gmtA is an essential gene. The lethal phenotype of the gmtA could be fully complemented by expressing an YFP-GmtA fusion protein from the endogenous gmtA promoter. Fluorescence studies revealed that, as in other fungal species, GmtA localized as punctate dots throughout the hyphal cytoplasm, representing Golgi bodies or Golgi equivalents. SrgC encodes a member of the Rab6/Ypt6 subfamily of secretion-related GTPases and is predicted to be required for the Golgi to vacuole transport. Loss of function of the srgC gene in A. niger resulted in strongly reduced growth and the inability to form conidiospores at 37°C and higher. Furthermore, the srgC disruption in the A. niger strain expressing the functional YFP-GmtA fusion protein led to an apparent 'disappearance' of the Golgi-like structures. The analysis suggests that SrgC has an important role in maintaining the integrity of Golgi-like structures in A. niger., (Copyright © 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Filamentous fungi have a high-capacity secretory system and are therefore widely exploited for the industrial production of native and heterologous proteins. However, in most cases, the yields of nonfungal proteins are significantly lower than those obtained for fungal proteins. One well-studied bottleneck appears to be the result of slow or aberrant folding of heterologous proteins in the ER during the early stages of secretion within the endoplasmic reticulum, leading to stress responses in the host, including the unfolded protein response (UPR). Most of the key elements constituting the signal transduction pathway of the UPR in Saccharomyces cerevisiae have been identified in filamentous fungi, including the central activation mechanism of the pathway, that is, the stress-induced splicing of an unconventional (nonspliceosomal) intron in orthologs of the HAC1 mRNA. This splicing event relieves a translational block in the HAC1 mRNA, allowing for the translation of the bZIP transcription factor Hac1p that regulates the expression of UPR target genes. The UPR is involved in regulating the folding, yield, and delivery of secretory proteins and that has consequences for fungal lifestyles, including virulence and biotechnology. The recent releases of genome sequences of several species of filamentous fungi and the availability of DNA arrays, GeneChips, and deep sequencing methodologies have provided an unprecedented resource for exploring expression profiles in response to secretion stresses. Furthermore, genome-wide investigation of translation profiles through polysome analyses is possible, and here, we outline methods for the use of such techniques with filamentous fungi and, principally, Aspergillus niger. We also describe methods for the batch and controlled cultivation of A. niger and for the replacement and study of its hacA gene, which provides either a UPR-deficient strain or a constitutively activated UPR strain for comparative analysis with its wild type. Although we focus on A. niger, the utility of the hacA-deletion strategy is also described for use in investigating the virulence of the plant pathogen Alternaria brassicicola., (Copyright © 2011 Elsevier Inc. All rights reserved.)