TRAPP structures reveal the big picture.

Two papers in this issue provide new structural insights into the "TRAnsport Protein Particle" (TRAPP) complexes, which play crucial roles in Golgi function. Both papers focus on TRAPPIII, which activates the Rab protein Ypt1 in yeast or the homologous Rab1 in metazoans. The structures illuminate how TRAPPIII specifically recognizes its Rab protein substrate. Joiner et al (2021) also describe a membrane-anchoring mechanism for yeast TRAPPIII, while Galindo et al (2021) characterize the large subunits that define metazoan TRAPPIII.

Membrane traffic within the Golgi apparatus.

Newly synthesized secretory cargo molecules pass through the Golgi apparatus while resident Golgi proteins remain in the organelle. However, the pathways of membrane traffic within the Golgi are still uncertain. Most of the available data can be accommodated by the cisternal maturation model, which postulates that Golgi cisternae form de novo, carry secretory cargoes forward and ultimately disappear. The entry face of the Golgi receives material that has been exported from transitional endoplasmic reticulum sites, and the exit face of the Golgi is intimately connected with endocytic compartments. These conserved features are enhanced by cell-type-specific elaborations such as tubular connections between mammalian Golgi cisternae. Key mechanistic questions remain about the formation and maturation of Golgi cisternae, the recycling of resident Golgi proteins, the origins of Golgi compartmental identity, the establishment of Golgi architecture, and the roles of Golgi structural elements in membrane traffic.

A photostable monomeric superfolder green fluorescent protein.

The green fluorescent protein (GFP) from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP variants show faster photobleaching. We now describe msGFP2, which retains monomeric superfolder properties while being as photostable as EGFP. msGFP2 contains modified N- and C-terminal peptides that are expected to reduce nonspecific interactions. Compared to EGFP and mEGFP, msGFP2 is less prone to disturbing the functions of certain partner proteins. For general-purpose protein tagging, msGFP2 may be the best available derivative of A. victoria GFP.

An improved secretion signal enhances the secretion of model proteins from Pichia pastoris.

BACKGROUND: Proteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal. RESULTS: We engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal. CONCLUSIONS: The improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins.

GenoLIB: a database of biological parts derived from a library of common plasmid features.

Synthetic biologists rely on databases of biological parts to design genetic devices and systems. The sequences and descriptions of genetic parts are often derived from features of previously described plasmids using ad hoc, error-prone and time-consuming curation processes because existing databases of plasmids and features are loosely organized. These databases often lack consistency in the way they identify and describe sequences. Furthermore, legacy bioinformatics file formats like GenBank do not provide enough information about the purpose of features. We have analyzed the annotations of a library of ∼2000 widely used plasmids to build a non-redundant database of plasmid features. We looked at the variability of plasmid features, their usage statistics and their distributions by feature type. We segmented the plasmid features by expression hosts. We derived a library of biological parts from the database of plasmid features. The library was formatted using the Synthetic Biology Open Language, an emerging standard developed to better organize libraries of genetic parts to facilitate synthetic biology workflows. As proof, the library was converted into GenoCAD grammar files to allow users to import and customize the library based on the needs of their research projects.

Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation.

Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1Δ cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in cisternal maturation kinetics. The arf1Δ mutation causes early Golgi cisternae to mature more slowly and less frequently, but does not alter the maturation of late Golgi cisternae. These changes quantitatively explain why late Golgi cisternae are fewer in number and correspondingly larger. With a stacked Golgi, similar changes in maturation kinetics could be used by the cell to modulate the number of cisternae per stack. Thus, the rates of processes that transform a maturing compartment can determine compartmental size and copy number.

Integrated self-organization of transitional ER and early Golgi compartments.

COPII coated vesicles bud from an ER domain termed the transitional ER (tER), but the mechanism that clusters COPII vesicles at tER sites is unknown. tER sites are closely associated with early Golgi or pre-Golgi structures, suggesting that the clustering of nascent COPII vesicles could be achieved by tethering to adjacent membranes. This model challenges the prevailing view that COPII vesicles are clustered by a scaffolding protein at the ER surface. Although Sec16 was proposed to serve as such a scaffolding protein, recent data suggest that rather than organizing COPII into higher-order structures, Sec16 acts at the level of individual COPII vesicles to regulate COPII turnover. A plausible synthesis is that tER sites are created by tethering to Golgi membranes and are regulated by Sec16. Meanwhile, the COPII vesicles that bud from tER sites are thought to nucleate new Golgi cisternae. Thus, an integrated self-organization process may generate tER-Golgi units.

Let there be order.

The Ringberg Colloquium on Self-Organization and Morphogenesis in Biological Systems took place between December 3-6, 2006 in a castle near Munich, Germany. Researchers from different areas of cell and developmental biology exchanged ideas about how biological systems are organized and dynamic at the same time. A dominant theme was that local interactions between molecules or cells can generate global order.

Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting.

BACKGROUND: Budding yeasts are often used to secrete foreign proteins, but the efficiency is variable. To identify roadblocks in the yeast secretory pathway, we used a monomeric superfolder GFP (msGFP) as a visual tracer in Saccharomyces cerevisiae and Pichia pastoris. RESULTS: One roadblock for msGFP secretion is translocation into the ER. Foreign proteins are typically fused to the bipartite α-factor secretion signal, which consists of the signal sequence followed by the pro region. The α-factor signal sequence directs posttranslational translocation. For msGFP, posttranslational translocation is inefficient with the α-factor signal sequence alone but is stimulated by the pro region. This requirement for the pro region can be bypassed by using the Ost1 signal sequence, which has been shown to direct cotranslational translocation. A hybrid secretion signal consisting of the Ost1 signal sequence followed by the α-factor pro region drives efficient translocation followed by rapid ER export. A second roadblock for msGFP secretion in S. cerevisiae occurs during exit from the Golgi, when some of the msGFP molecules are diverted to the vacuole. Deletion of the sorting receptor Vps10 prevents vacuolar targeting of msGFP at the expense of missorting vacuolar hydrolases such as carboxypeptidase Y (CPY) to the culture medium. However, a truncation of Vps10 blocks vacuolar targeting of msGFP while permitting CPY to be sorted normally. CONCLUSIONS: With budding yeasts, if the secretion or processing of a foreign protein is poor, we recommend two options. First, use the Ost1 signal sequence to achieve efficient entry into the secretory pathway while avoiding the processing issues associated with the α-factor pro region. Second, truncate Vps10 to suppress diversion to the vacuole. These insights obtained with msGFP highlight the value of applying cell biological methods to study yeast secretion.

Yellow and oxidation-resistant derivatives of a monomeric superfolder GFP.

Fluorescent proteins (FPs) are essential tools in biology. The utility of FPs depends on their brightness, photostability, efficient folding, monomeric state, and compatibility with different cellular environments. Despite the proliferation of available FPs, derivatives of the originally identified Aequorea victoria GFP often show superior behavior as fusion tags. We recently generated msGFP2, an optimized monomeric superfolder variant of A. victoria GFP. Here, we describe two derivatives of msGFP2. The monomeric variant msYFP2 is a yellow superfolder FP with high photostability. The monomeric variant moxGFP2 lacks cysteines but retains significant folding stability, so it works well in the lumen of the secretory pathway. These new FPs are useful for common imaging applications.

A three-stage model of Golgi structure and function.

The Golgi apparatus contains multiple classes of cisternae that differ in structure, composition, and function, but there is no consensus about the number and definition of these classes. A useful way to classify Golgi cisternae is according to the trafficking pathways by which the cisternae import and export components. By this criterion, we propose that Golgi cisternae can be divided into three classes that correspond to functional stages of maturation. First, cisternae at the cisternal assembly stage receive COPII vesicles from the ER and recycle components to the ER in COPI vesicles. At this stage, new cisternae are generated. Second, cisternae at the carbohydrate synthesis stage exchange material with one another via COPI vesicles. At this stage, most of the glycosylation and polysaccharide synthesis reactions occur. Third, cisternae at the carrier formation stage produce clathrin-coated vesicles and exchange material with endosomes. At this stage, biosynthetic cargo proteins are packaged into various transport carriers, and the cisternae ultimately disassemble. Discrete transitions occur as a cisterna matures from one stage to the next. Within each stage, the structure and composition of a cisterna can evolve, but the trafficking pathways remain unchanged. This model offers a unified framework for understanding the properties of the Golgi in diverse organisms.

The yeast GRASP Grh1 colocalizes with COPII and is dispensable for organizing the secretory pathway.

In mammalian cells, the 'Golgi reassembly and stacking protein' (GRASP) family has been implicated in Golgi stacking, but the broader functions of GRASP proteins are still unclear. The yeast Saccharomyces cerevisiae contains a single non-essential GRASP homolog called Grh1. However, Golgi cisternae in S. cerevisiae are not organized into stacks, so a possible structural role for Grh1 has been difficult to test. Here, we examined the localization and function of Grh1 in S. cerevisiae and in the related yeast Pichia pastoris, which has stacked Golgi cisternae. In agreement with earlier studies indicating that Grh1 interacts with coat protein II (COPII) vesicle coat proteins, we find that Grh1 colocalizes with COPII at transitional endoplasmic reticulum (tER) sites in both yeasts. Deletion of P. pastoris Grh1 had no obvious effect on the structure of tER-Golgi units. To test the role of S. cerevisiae Grh1, we exploited the observation that inhibiting ER export in S. cerevisiae generates enlarged tER sites that are often associated with the cis Golgi. This tER-Golgi association was preserved in the absence of Grh1. The combined data suggest that Grh1 acts early in the secretory pathway, but is dispensable for the organization of secretory compartments.

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.

Clathrin adaptors mediate two sequential pathways of intra-Golgi recycling.

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.

A noncytotoxic DsRed variant for whole-cell labeling.

A common application of fluorescent proteins is to label whole cells, but many RFPs are cytotoxic when used with standard high-level expression systems. We engineered a rapidly maturing tetrameric fluorescent protein called DsRed-Express2 that has minimal cytotoxicity. DsRed-Express2 exhibits strong and stable expression in bacterial and mammalian cells, and it outperforms other available RFPs with regard to photostability and phototoxicity.

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.

Bioreactor-scale cell performance and protein production can be substantially increased by using a secretion signal that drives co-translational translocation in Pichia pastoris.

Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for production of heterologous proteins of biotechnological interest, many of them extracellular. The protein secretion pathway has been recognized as a limiting process in which many roadblocks have been pinpointed. Recently, we have identified a bottleneck at the ER translocation level. In earlier exploratory studies, this limitation could be largely overcome by using an improved chimeric secretion signal to drive proteins through the co-translational translocation pathway. Here, we have further tested at bioreactor scale the improved secretion signal consisting of the pre-Ost1 signal sequence, which drives proteins through co-translational translocation, followed by the pro region from the secretion signal of the Saccharomyces cerevisiae α-factor mating pheromone. For comparison, the commonly used full-length α-factor secretion signal, which drives proteins through post-translational translocation, was tested. These two secretion signals were fused to three different model proteins: the tetrameric red fluorescent protein E2-Crimson, which can be used to visualize roadblocks in the secretory pathway; the lipase 2 from Bacillus thermocatenulatus (BTL2); and the Rhizopus oryzae lipase (ROL). All strains were tested in batch cultivation to study the different growth parameters obtained. The strains carrying the improved secretion signal showed increased final production of the proteins of interest. Interestingly, they were able to grow at significantly higher maximum specific growth rates than their counterparts carrying the conventional secretion signal. These results were corroborated in a 5 L fed-batch cultivation, where the final product concentration and volumetric productivity were also shown to be improved.

Acetyl-CoA flux from the cytosol to the ER regulates engagement and quality of the secretory pathway.

Nε-lysine acetylation in the ER is an essential component of the quality control machinery. ER acetylation is ensured by a membrane transporter, AT-1/SLC33A1, which translocates cytosolic acetyl-CoA into the ER lumen, and two acetyltransferases, ATase1 and ATase2, which acetylate nascent polypeptides within the ER lumen. Dysfunctional AT-1, as caused by gene mutation or duplication events, results in severe disease phenotypes. Here, we used two models of AT-1 dysregulation to investigate dynamics of the secretory pathway: AT-1 sTg, a model of systemic AT-1 overexpression, and AT-1S113R/+, a model of AT-1 haploinsufficiency. The animals displayed reorganization of the ER, ERGIC, and Golgi apparatus. In particular, AT-1 sTg animals displayed a marked delay in Golgi-to-plasma membrane protein trafficking, significant alterations in Golgi-based N-glycan modification, and a marked expansion of the lysosomal network. Collectively our results indicate that AT-1 is essential to maintain proper organization and engagement of the secretory pathway.

A General Method to Improve Fluorophores Using Deuterated Auxochromes.

Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways-thereby increasing the brightness and photostability of fluorophores-are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.