Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris.
It was recently reported that co-expression of the proalpha1(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J. 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only approximately 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human proalpha1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical proalpha1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments. The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule.
Keizer-Gunnink
I
I
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Biological Center, University of Groningen, Kerklaan 30, 9751 NN, Haren, Netherlands.
Vuorela
A
A
Myllyharju
J
J
Pihlajaniemi
T
T
Kivirikko
K I
KI
Veenhuis
M
M
D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D010675:Pheromones; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011347:Procollagen / Q000235:genetics / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D008074:Lipoproteins; C095063:MFA2 protein, S cerevisiae; D010675:Pheromones; D011347:Procollagen; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D011393:Procollagen-Proline Dioxygenase