Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum

We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation.The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.Though the secretory pathway is organized similarly in yeast and in other eukaryotic organisms, not all proteins of higher eukaryotes can be efficiently secreted from yeast cells. Secretion of some recombinant or mutant proteins is affected by their improper folding in the yeast endoplasmic reticulum (ER) [1,2] and rapid degradation of misfolded molecules by cytosolic proteasome complex after retrograde translocation from the lumenal to cytoplasmic face of the ER (ER associated protein degradation, ERAD) [3,4]. Alternatively, misfolded proteins can be sorted from the late Golgi to vacuole, yeast homologue of lysosome, and degraded by the vacuolar protease complex [5,6]. The recognition and retention of misfolded proteins in the ER is carried out by the "ER quality control" (ERQC) machinery. Saccharomyces cerevisiae differs by the organization of ERQC not only from higher eukaryotes, but also from Schizosaccharomyces pombe. However, in spite of these species-specific differences, the ERQC involves processing of the N-linked oligosaccharides in all eukaryotes reflecting importance of N-glycosylation for protein folding in vivo [for review, see [7]].Human urok