Jeremy C. Simpson

Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing.

As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N‐ and C‐terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of >100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For >80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum.

The cytosolic coat-protein complex COP-I interacts with cytoplasmic ‘retrieval’ signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.

Ricin A chain utilises the endoplasmic reticulum‐associated protein degradation pathway to enter the cytosol of yeast

Cytotoxic proteins such as ricin A chain (RTA) have target substrates in the cytosol and therefore have to reach this cellular compartment in order to act. RTA is thought to translocate into the cytosol from the lumen of the endoplasmic reticulum (ER), although how it traverses the ER membrane has not been established. Using yeast mutants defective in various aspects of the ER‐associated protein degradation (ERAD) pathway, we show that RTA introduced into the yeast ER subverts this pathway to enter the cytosol via the Sec61p translocon. A significant proportion of the exported RTA avoided proteasomal degradation. These data are consistent with the contention that the RTA component from ricin endocytosed by mammalian cells may likewise exploit ERAD to translocate into the cytosol.

Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway

The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.

A role for the small GTPase Rab21 in the early endocytic pathway.

Rab proteins comprise a family of monomeric GTPases that control cellular membrane traffic. Rab21 is a poorly characterised member with no known function. Human Rab21 cDNA from K562 cells was subcloned into GFP expression vectors to generate Rab21 and Rab21 mutants defective in either GTP hydrolysis (Rab21 Q78L) or binding (Rab21 T33N) for transfection studies in HeLa cells. Confocal fluorescence microscopy and ultrastructural studies revealed Rab21 to be predominantly localised to the early endocytic pathway, on vesicles containing earlyendosomal antigen 1 EEA1, transferrin receptor and internalised ligands. EEA1 was localised to enlarged endosomes in Rab21 wild-type expressing cells but the GTP hydrolysis and GDP binding mutants had unique phenotypes labelling tubular reticular structures and the trans-Golgi network, respectively. Early endosome localisation for Rab21 was confirmed in a hepatoma cell line that allowed analysis of the subcellular distribution of the endogenous protein. Comparison of the localisation of Rab21 with other Rabs revealed extensive colocalisation with early endocytic variants Rab4, Rab5, Rab17 and Rab22 but much less overlap with those associated with late endosomes, recycling endosomes and the early secretory pathway. Cells expressing Rab21 T33N had defects in endocytosis of transferrin and epidermal growth factor and failed to effectively deliver the latter ligand to late endosomes and lysosomes for degradation. Collectively, our data provide the first characterisation of Rab21 function in early endosome dynamics.

Analysis of Intraviral Protein-Protein Interactions of the SARS Coronavirus ORFeome

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.

A microscope-based screening platform for large-scale functional protein analysis in intact cells.

A modular microscope‐based screening platform, with applications in large‐scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome‐wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large‐scale, cell‐based functional proteomic projects.

Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells

Imaging techniques such as immunofluorescence (IF) and the expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Here we report a systematic analysis of >500 human proteins comparing the localizations obtained in live versus fixed cells using FPs and IF, respectively. We identify systematic discrepancies between IF and FPs as well as between FP tagging at the N and C termini. The analysis shows that for 80% of the proteins, IF and FPs yield the same subcellular distribution, and the locations of 250 previously unlocalized proteins were determined by the overlap between the two methods. Approximately 60% of proteins localize to multiple organelles for both methods, indicating a complex subcellular protein organization. These results show that both IF and FP tagging are reliable techniques and demonstrate the usefulness of an integrative approach for a complete investigation of the subcellular human proteome.

Rab18 and Rab43 have key roles in ER-Golgi trafficking

Rabs and Arfs/Arls are Ras-related small GTPases of particular relevance to membrane trafficking. It is thought that these proteins regulate specific pathways through interactions with coat, motor, tether and SNARE proteins. We screened a comprehensive list of Arf/Arl/Rab proteins, previously identified on purified Golgi membranes by a proteomics approach (37 in total), for Golgi or intra-Golgi localization, dominant-negative and overexpression phenotypes. Further analysis of two of these proteins, Rab18 and Rab43, strongly indicated roles in ER-Golgi trafficking. Rab43-T32N redistributed Golgi elements to ER exit sites without blocking trafficking of the secretory marker VSVG-GFP from ER to cell surface. Wild-type Rab43 redistributes the p150Glued subunit of dynactin, consistent with a specific role in regulating association of pre-Golgi intermediates with microtubules. Overexpression of wild-type GFP-Rab18 or incubation with any of three siRNAs directed against Rab18 severely disrupts the Golgi complex and reduces secretion of VSVG. Rab18 mutants specifically enhance retrograde Golgi-ER transport of the COPI-independent cargo β-1,4-galactosyltransferase (Galtase)-YFP but not the COPI-dependent cargo p58-YFP from the Golgi to ER in a photobleach assay. Rab18-S22N also potentiated brefeldin-A-induced ER-Golgi fusion. This study is the first comprehensive application of large-scale proteomics to the cell biology of small GTPases of the secretory pathway.

RNA Bacteriophage Capsid-Mediated Drug Delivery and Epitope Presentation

Objective: To use our knowledge of the three-dimensional structure and self-assembly mechanism of RNA bacteriophage capsids to develop novel virus-like particles (VLPs) for drug delivery and epitope presentation. Methods: Site-directed mutagenesis of a recombinant MS2 coat protein expression construct has been used to generate translational fusions encompassing short epitope sequences. These chimeric proteins still self-assemble in vivo into T = 3 shells with the foreign epitope in an accessible location. Covalent conjugation has also been used to generate RNA stem-loops attached to the toxin, ricin A chain, or to nucleotide-based drugs, that are still capable of stimulating self-assembly of the capsid in vitro. These packaged drugs can then be directed to specific cells in culture by further covalent decoration of the capsids with targeting molecules. Results: Chimeric VLPs are strongly immunogenic when carrying either B or T cell epitopes, the latter generating cytokine profiles consistent with memory responses. Immune responses to the underlying phage epitopes appear to be proportional to the area of the phage surface accessible. Phage shells effectively protect nucleic acid-based drugs and, for the toxin construct, make cell-specific delivery systems with LD50 values in culture sub-nanomolar. Conclusion: VLP technology has potential for therapeutic and prophylactic intervention in disease.