Cemal Gurkan

Structure of the Sec13/31 COPII coat cage

Endomembranes of eukaryotic cells are dynamic structures that are in continuous communication through the activity of specialized cellular machineries, such as the coat protein complex II (COPII), which mediates cargo export from the endoplasmic reticulum (ER). COPII consists of the Sar1 GTPase, Sec23 and Sec24 (Sec23/24), where Sec23 is a Sar1-specific GTPase-activating protein and Sec24 functions in cargo selection, and Sec13 and Sec31 (Sec13/31), which has a structural role. Whereas recent results have shown that Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles, we now show that Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24. We present a three-dimensional reconstruction of these Sec13/31 cages at 30 Å resolution using cryo-electron microscopy and single particle analysis. These results reveal a novel cuboctahedron geometry with the potential to form a flexible lattice and to generate a diverse range of containers. Our data are consistent with a model for COPII coat complex assembly in which Sec23/24 has a non-structural role as a multivalent ligand localizing the self-assembly of Sec13/31 to form a cage lattice driving ER cargo export.

Structural basis for cargo regulation of COPII coat assembly.

Using cryo-electron microscopy, we have solved the structure of an icosidodecahedral COPII coat involved in cargo export from the endoplasmic reticulum (ER) coassembled from purified cargo adaptor Sec23-24 and Sec13-31 lattice-forming complexes. The coat structure shows a tetrameric assembly of the Sec23-24 adaptor layer that is well positioned beneath the vertices and edges of the Sec13-31 lattice. Fitting the known crystal structures of the COPII proteins into the density map reveals a flexible hinge region stemming from interactions between WD40 beta-propeller domains present in Sec13 and Sec31 at the vertices. The structure shows that the hinge region can direct geometric cage expansion to accommodate a wide range of bulky cargo, including procollagen and chylomicrons, that is sensitive to adaptor function in inherited disease. The COPII coat structure leads us to propose a mechanism by which cargo drives cage assembly and membrane curvature for budding from the ER.

The COPII cage: unifying principles of vesicle coat assembly

Communication between compartments of the exocytic and endocytic pathways in eukaryotic cells involves transport carriers — vesicles and tubules — that mediate the vectorial movement of cargo. Recent studies of transport-carrier formation in the early secretory pathway have provided new insights into the mechanisms of cargo selection by coat protein complex-II (COPII) adaptor proteins, the construction of cage-protein scaffolds and fission. These studies are beginning to produce a unifying molecular and structural model of coat function in the formation and fission of vesicles and tubules in endomembrane traffic.

An Evolutionary Perspective on Eukaryotic Membrane Trafficking

The eukaryotic cell is defined by a complex set of sub-cellular compartments that include endomembrane systems making up the exocytic and endocytic trafficking pathways. Current evidence suggests that both the function and communication between these compartments are regulated by distinct families of proteins that direct membrane fission, targeting and fusion. These families include coat protein complexes (CPCs) involved in vesicle formation/fission, Rab GTPases involved in vesicle targeting, and soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs) involved in vesicle fusion. The origins of these gene families and their individual contributions to the evolutionary specialization of the membrane architectures of lower and higher eukaryotes are now better understood with the advent of powerful phylogenetic, structural and systems biology tools. Herein, we provide a perspective that suggests that while the core CPC and SNARE machineries have diversified modestly in the course of eukaryotic evolution, the Rab GTPase family expanded substantially to emerge as a key driving force in endomembrane specialization. The Rab GTPases appear to have provided the foundation for the intricate membrane architectures ranging from those requisite for the distinct amoebic life cycle stage of uni-cellular organisms such as the parasitic protozoa to the highly specialized tissue and cell type-specific endomembranes of multi-cellular eukaryotes. We propose that Rab-centric interaction networks orchestrate the divergent activities of fission and fusion through their capacity to control the sequential assembly of protein complexes that mediate endomembrane structure and communication.

Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin.

Quantitative Proteomic Profiling of Matched Normal and Tumor Breast Tissues

Proteomic analysis of breast cancer tissue has proven difficult due to its inherent histological complexity. This pilot study presents preliminary evidence for the ability to differentiate adenoma and invasive carcinoma by measuring changes in proteomic profile of matched normal and disease tissues. A dual lysis buffer method was used to maximize protein extraction from each biopsy, proteins digested with trypsin, and the resulting peptides iTRAQ labeled. After combining, the peptide mixtures they were separated using preparative IEF followed by RP nanoHPLC. Following MALDI MS/MS and database searching, identified proteins were combined into a nonredundant list of 481 proteins with associated normal/tumor iTRAQ ratios for each patient. Proteins were categorized by location as blood, extracellular, and cellular, and the iTRAQ ratios were normalized to enable comparison between patients. Of those proteins significantly changed (upper or lower quartile) between matched normal and disease tissues, those from two invasive carcinoma patients had >50% in common with each other but <22% in common with an adenoma patient. In invasive carcinoma patients, several cellular and extracellular proteins that were significantly increased (Periostin, Small breast epithelial mucin) or decreased (Kinectin) have previously been associated with breast cancer, thereby supporting this approach for a larger disease-stage characterization effort.

High-level production in Pichia pastoris of an anti-p185HER-2 single-chain antibody fragment using an alternative secretion expression vector

The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the recombinant production of a wide variety of proteins. Initial success with this system was greatly facilitated by the development of versatile expression vectors that were almost exclusively based on the strong, tightly regulated promoter of the P. pastoris major alcohol oxidase gene (AOX1). For example, pIB4 is an Escherichia coli–P. pastoris shuttle vector that also uses the AOX1 promoter to allow intracellular expression of endogenous and foreign genes in the latter organism. Since the eukaryotic advantages of P. pastoris would be best harnessed through the secretory targeting of the recombinant proteins, we modified the pIB4 vector by adding the Saccharomyces cerevisiae α‐factor secretion signal immediately upstream of its multiple cloning site. Here we describe the construction of this modified vector, pIB4α, and its successful use for the high‐level expression and secretion of a functional single‐chain antibody fragment (scFv), C6.5, which targets p185HER‐2, a cell‐surface glycoprotein overexpressed in about 30% of human breast and ovarian cancers. The PCR strategy used for the subcloning of the C6.5 construct into pIB4α also introduced a short DNA sequence coding for a C‐terminal hexahistidine tag, which allowed subsequent purification of the secreted scFv, by immobilized‐metal‐affinity chromatography, to a yield of 70 mg·l−1 of shake‐flask culture. In conclusion, our results suggest that the secretion expression vector pIB4α not only complements the original pIB4 vector for intracellular expression in P. pastoris, but might also constitute an attractive alternative to the commercially available secretion expression vectors.

52 additional reference population samples for the 55 AISNP panel

Ancestry inference for a person using a panel of SNPs depends on the variation of frequencies of those SNPs around the world and the amount of reference data available for calculation/comparison. The Kidd Lab panel of 55 AISNPs has been incorporated in commercial kits by both Life Technologies and Illumina for massively parallel sequencing. Therefore, a larger set of reference populations will be useful for researchers using those kits. We have added reference population allele frequencies for 52 population samples to the 73 previously entered so that there are now allele frequencies publicly available in ALFRED and FROG-kb for a total of 125 population samples.

Mise en Place–This Bud's for the Golgi

Selective cargo export from the endoplasmic reticulum is brought about by the budding of COPII vesicles. While the main structural components of the COPII coat have been identified and characterized, the regulatory event(s) promoting COPII vesicle biogenesis and cargo selection still remains largely unknown. New data by Glick and colleagues suggest that Sec12 and COPII function may be downstream of important early events coordinated by transitional ER (tER) exit sites.

Expression of the Bacillus thuringiensis Cyt2Aa1 toxin in Pichia pastoris using a synthetic gene construct

Bacillus thuringiensis δ‐endotoxins are membrane‐active, pore‐forming proteins with highly specific insecticidal activities. In addition to a well‐established role in the biological control of a wide variety of crop pests and disease vectors, these toxins also have great potential for the development of anti‐tumour agents called immunotoxins (ITs), chimaeric molecules consisting of a cell‐binding ligand coupled to a toxin or its subunits. The ultimate goal of our study was the recombinant production of such ITs based on the Cyt2Aa1 toxin from B. thuringiensis subspecies kyushuensis. We explored the use of Pichia pastoris for recombinant IT production because earlier attempts in our laboratory using the Escherichia coli expression system or various chemical conjugation strategies yielded only low levels of functional product. However, our initial attempts were not successful because the A+T‐rich bacterial cyt2Aa1 gene contained fortuitous polyadenylation sites, causing premature transcription termination in this yeast. Accordingly, we designed and constructed a synthetic cyt2Aa1 gene (syncyt2Aa1) optimized for heterologous expression in P. pastoris. This was achieved by increasing the overall G+C content of the bacterial cyt2Aa1 while changing its codon usage to that preferred by the methylotrophic yeast. Here we describe in detail the design, synthesis and requisite PCR repair of syncyt2Aa1, then present analyses of recombinant Cyt2Aa1 expression in P. pastoris using this synthetic gene. Following the results presented in this paper, the syncyt2Aa1 gene was also successfully used for the recombinant production of a Cyt2Aa1‐based IT in the same expression host [C. Gurkan and D. J. Ellar (2003) Protein Expression Purification 29, 103–116].