Brooke Bevis

Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed)

The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with 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. An asparagine-to-glutamine substitution at position 42 greatly accelerates the maturation of DsRed, but also increases the level of green emission. Additional amino acid substitutions suppress this green emission while further accelerating the maturation. To enhance the solubility of DsRed, we reduced the net charge near the N terminus of the protein. The optimized DsRed variants yield bright fluorescence even in rapidly growing organisms such as yeast.

Golgi maturation visualized in living yeast.

The Golgi apparatus is composed of biochemically distinct early (cis, medial) and late (trans, TGN) cisternae. There is debate about the nature of these cisternae. The stable compartments model predicts that each cisterna is a long-lived structure that retains a characteristic set of Golgi-resident proteins. In this view, secretory cargo proteins are transported by vesicles from one cisterna to the next. The cisternal maturation model predicts that each cisterna is a transient structure that matures from early to late by acquiring and then losing specific Golgi-resident proteins. In this view, secretory cargo proteins traverse the Golgi by remaining within the maturing cisternae. Various observations have been interpreted as supporting one or the other mechanism. Here we provide a direct test of the two models using three-dimensional time-lapse fluorescence microscopy of the yeast Saccharomyces cerevisiae. This approach reveals that individual cisternae mature, and do so at a consistent rate. In parallel, we used pulse–chase analysis to measure the transport of two secretory cargo proteins. The rate of cisternal maturation matches the rate of protein transport through the secretory pathway, suggesting that cisternal maturation can account for the kinetics of secretory traffic.

De novo formation of transitional ER sites and Golgi structures in Pichia pastoris.

Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER. Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi structures in the budding yeast Pichia pastoris. The biogenesis of tER sites is tightly linked to the biogenesis of Golgi, and both compartments can apparently form de novo. tER sites often fuse with one another, but they maintain a consistent average size through shrinkage after fusion and growth after de novo formation. Golgi dynamics are similar, although late Golgi elements often move away from tER sites towards regions of polarized growth. Our results can be explained by assuming that tER sites give rise to Golgi cisternae that continually mature.

The Assembly of Amyloidogenic Yeast Sup35 as Assessed by Scanning (Atomic) Force Microscopy: An Analogy to Linear Colloidal Aggregation?

Amyloidosis is a class of diseases caused by protein aggregation and deposition in various tissues and organs. In this paper, a yeast amyloid-forming protein Sup35 was used as a model for understanding amyloid fiber formation. The dynamics of amyloid formation by Sup35 were studied with scanning force microscopy. We found that: 1) the assembly of Sup35 fibers begins with individual NM peptides that aggregate to form large beads or nucleation units which, in turn, form dimers, trimers, tetramers and longer linear assemblies appearing as a string of beads; 2) the morphology of the linear assemblies differ; and 3) fiber assembly suggests an analogy to the aggregation of colloidal particles. A dipole assembly model is proposed based on this analogy that will allow further experimental testing.

Isolation of Pichia pastoris genes involved in ER-to-Golgi transport

Pichia pastoris has discrete transitional ER sites and coherent Golgi stacks, making this yeast an ideal system for studying the organization of the early secretory pathway. To provide molecular tools for this endeavour, we isolated P. pastoris homologues of the SEC12, SEC13, SEC17, SEC18 and SAR1 genes. The P. pastoris SEC12, SEC13, SEC17 and SEC18 genes were shown to complement the corresponding S. cerevisiae mutants. The SEC17 and SAR1 genes contain introns at the same relative positions in both P. pastoris and S. cerevisiae, whereas the SEC13 gene contains an intron in P. pastoris but not in S. cerevisiae. Intron structure is similar in the two yeasts, although the favoured 5′ splice sequence appears to be GTAAGT in P. pastoris vs. GTATGT in S. cerevisiae. The predicted amino acid sequences of Sec13p, Sec17p, Sec18p and Sar1p show strong conservation in the two yeasts. By contrast, the predicted lumenal domain of Sec12p is much larger in P. pastoris, suggesting that this domain may help localize Sec12p to transitional ER sites. A comparison of the SEC12 loci in various budding yeasts indicates that the SEC12‐related gene SED4 is probably unique to the Saccharomyces lineage. GenBank Accession Nos are: SEC12, AF216960; SEC13, AF242186; SEC17, AF216957; SEC18, AF216958; SAR1, AF216959; ACT1, AF216956. Copyright

Morphogenesis signaling components influence cell cycle regulation by cyclin dependent kinase

BackgroundThe yeast cell cycle is largely controlled by the cyclin-dependent kinase (CDK) Cdc28. Recent evidence suggests that both CDK complex stability as well as function during mitosis is determined by precise regulation of Swe1, a CDK inhibitory kinase and cyclin binding partner. A model of mitotic progression has been provided by study of filamentous yeast. When facing nutrient-limited conditions, Ras2-mediated PKA and MAPK signaling cascades induce a switch from round to filamentous morphology resulting in delayed mitotic progression.ResultsTo delineate how the dimorphic switch contributes to cell cycle regulation, temperature sensitive cdc28 mutants exhibiting constitutive filamentation were subjected to epistasis analyses with RAS2 signaling effectors. It was found that Swe1-mediated inhibitory tyrosine phosphorylation of Cdc28 during filamentous growth is in part mediated by Ras2 activation of PKA, but not Kss1-MAPK, signaling. This pathway is further influenced by Cks1, a conserved CDK-binding partner of elusive function with multiple proposed roles in CDK activation, transcriptional regulation and ubiquitin-mediated proteasome degradation.ConclusionThe dynamic balance between Cks1- and Swe1-dependent regulation of Cdc28 and, thereby, the timing of mitosis during yeast dimorphism is regulated in part by Ras2/cAMP-mediated PKA signaling, a key pathway controlling filamentous growth.