Overproduction of BiP negatively affects the secretion of Aspergillus niger glucose oxidase by the yeast Hansenula polymorpha.
We have cloned the Hansenula polymorpha BIP gene from genomic DNA using a PCR-based strategy. H. polymorpha BIP encodes a protein of 665 amino acids, which shows very high homology to Saccharomvces cerevisiae KAR2p. KAR2p belongs to the Hsp70 family of molecular chaperones and resides in the endoplasmic reticulum (ER)-lumen. H. polymorpha BiP contains a putative N-terminal signal sequence of 30 amino acids together with the conserved -HDEL sequence, the typical ER retention signal, at the extreme C-terminus. We have analysed the effect of BIP overexpression, placing the gene under control of the strong alcohol oxidase promoter (P(MOX)) on the secretion of artificially produced Aspergillus niger glucose oxidase (GOX) by H. polymorpha. BiP overproduction did not lead to any growth defects of the cells; at the subcellular level, proliferation of ER-like vesicles was observed. However, artificially enhanced BiP levels strongly affected GOX secretion and led to accumulation of this protein in the ER-like vesicles. This was not simply due to the high BiP overproduction, because it was also observed under conditions of low P(MOX) induction during growth of cells on glycerol. Vacuolar carboxypeptidase Y was properly sorted to its target organelle in the BiP overproducing strains.
van der Heide
M
M
Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands.
Hollenberg
C P
CP
van der Klei
I J
IJ
Veenhuis
M
M
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005949:Glucose Oxidase / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000502:physiology; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C060153:KAR2 protein, yeast; D011994:Recombinant Proteins; D005949:Glucose Oxidase