Biogenesis and dynamics of the early secretory pathway in Pichia pastoris.

摘要

Different models for Golgi function profoundly affect our concept of the Golgi and its relationship to other organelles in the secretory pathway. The cisternal maturation model for Golgi function represents the Golgi as an outgrowth of the transitional ER (tER), an ER subdomain that produces COPII transport vesicles. We have tested predictions of the cisternal maturation model by investigating the relationship between the tER and the Golgi in budding yeasts. In Pichia pastoris, Golgi stacks are adjacent to discrete tER sites, whereas in Saccharomyces cerevisiae, the entire ER network appears to function as tER. We propose that the discrete tER sites in P. pastoris generate coherent Golgi stacks, while the delocalized tER in S. cerevisiae generates a dispersed Golgi. This correlation between Golgi structure and tER organization is consistent with the cisternal maturation model.; The link between the tER and the Golgi prompted us to investigate the biogenesis and dynamics of these structures using two-color confocal video microscopy. The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with the green fluorescent protein (GFP), but wild-type DsRed has several drawbacks, including slow chromophore maturation and poor solubility. To overcome the slow maturation, we used random and directed mutagenesis to create DsRed variants that mature 10–15 times faster than the wild-type protein. The optimized DsRed variants yield bright fluorescence even in rapidly growing organisms such as yeast and are suitable for two-color microscopy experiments with GFP.; Using one- and two-color confocal video microscopy, we characterized the relationship between tER dynamics and Golgi dynamics in P. pastoris . tER sites can form de novo and fuse with one another. These observations suggest a self-association model for tER formation and maintenance. Golgi structures also appear to form de novo and fuse with other Golgi structures. Interestingly, tER formation and Golgi formation are closely linked, with new Golgi structures arising next to recently formed tER sites. This observation suggests that the Golgi grows out of the tER. Taken together, our findings are consistent with the idea that the tER is the birthplace of the Golgi.