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 proal(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 similar to 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human pro alpha 1(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 pro alpha 1(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. (C) 2000 Elsevier Science B.V./International Society of Matrix Biology. All rights reserved

Transposon mutations in the flagella biosynthetic pathway of the solvent-tolerant Pseudomonas putida S12 result in a decreased expression of solvent efflux genes

Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMOD-KmO mutagenesis system. These mutants were unable to grow in the presence of octanol and toluene. By cloning the region flanking the transposon insertion point a partial sequence of the interrupted genes was determined. Comparison of the deduced amino acid sequences with a protein database revealed the following interrupted putative gene products: organic solvent efflux proteins SrpA and SrpB. the flagellar structural proteins FlgK, FlaG, Flif. FliC. and FliH, the transcriptional activator FleQ, the alternative RNA polymerase sigma Factor RpoN, and the flagellum-specific RNA polymerase sigma factor FliA (RpoF). The transposon mutants, except for the organic solvent afflux mutants, were nonmotile as determined by a swarm assay and the formation of the flagellum was totally impaired. Expression studies with a sri, Promoter probe showed a decreased expression of the SrpABC efflux pump in the nonmotile mutants. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Ail rights reserved

Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting

We have been studying protein components that function in the cytoplasm to vacuole targeting (Cvt) pathway and the overlapping process of macroautophagy. The Vac8 and Apg13 proteins are required for the import of aminopeptidase I (API) through the Cvt pathway. We have identified a protein-protein interaction between Vac8p and Apg13p by both two-hybrid and co-immunoprecipitation analysis. Subcellular fractionation of API indicates that Vac8p and Apg13p are involved in the vesicle formation step of the Cvt pathway. Kinetic analysis of the Cvt pathway and autophagy indicates that, although Vac8p is essential for Cvt transport, it is less important for autophagy, In vivo phosphorylation experiments demonstrate that both Vac8p and Apg13p are phosphorylated proteins, and Apg13p phosphorylation is regulated by changing nutrient conditions. Although Apg13p interacts with the serine/threonine kinase Apg1p, this protein is not required for phosphorylation of either Vac8p or Apg13p, Subcellular fractionation experiments indicate that Apg13p and a fraction of Apg1p are membrane-associated, Vac8p and Apg13p may be part of a larger protein complex that includes Apg1p and additional interacting proteins. Together, these components may form a protein complex that regulates the conversion between Cvt transport and autophagy in response to changing nutrient conditions