Isoelectric focusing technology quantifies protein signaling in 25 cells.

A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High-resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.

Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen.

ER-associated degradation (ERAD) of glycoproteins depends on dual recognition of protein misfolding and remodeling of the substrate's N-linked glycans. After recognition, substrates are retrotranslocated to the cytosol for proteasomal degradation. To explore the directionality of this process, we fused a highly stable protein, DHFR, to the N or C terminus of the soluble ERAD substrate CPY* in yeast. Degradation of the C-terminal CPY*-DHFR fusion is markedly slowed and is accompanied by DHFR release in the ER lumen. Thus, folded lumenal domains can impede protein retrotranslocation. The ER lumenal protein Yos9p is required for both release of DHFR and degradation of multiple ERAD substrates. Yos9p forms a complex with substrates and has a sugar binding pocket that is essential for its ERAD function. Nonetheless, substrate recognition persists even when the sugar binding site is mutated or CPY* is unglycosylated. These and other considerations suggest that Yos9p plays a critical role in the bipartite recognition of terminally misfolded glycoproteins.

Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.

We present a strategy for generating and analyzing comprehensive genetic-interaction maps, termed E-MAPs (epistatic miniarray profiles), comprising quantitative measures of aggravating or alleviating interactions between gene pairs. Crucial to the interpretation of E-MAPs is their high-density nature made possible by focusing on logically connected gene subsets and including essential genes. Described here is the analysis of an E-MAP of genes acting in the yeast early secretory pathway. Hierarchical clustering, together with novel analytical strategies and experimental verification, revealed or clarified the role of many proteins involved in extensively studied processes such as sphingolipid metabolism and retention of HDEL proteins. At a broader level, analysis of the E-MAP delineated pathway organization and components of physical complexes and illustrated the interconnection between the various secretory processes. Extension of this strategy to other logically connected gene subsets in yeast and higher eukaryotes should provide critical insights into the functional/organizational principles of biological systems.

Functional overlap between retinitis pigmentosa 2 protein and the tubulin-specific chaperone cofactor C.

Mutations in the X-linked retinitis pigmentosa 2 gene cause progressive degeneration of photoreceptor cells. The retinitis pigmentosa 2 protein (RP2) is similar in sequence to the tubulin-specific chaperone cofactor C. Together with cofactors D and E, cofactor C stimulates the GTPase activity of native tubulin, a reaction regulated by ADP-ribosylation factor-like 2 protein. Here we show that in the presence of cofactor D, RP2 protein also stimulates the GTPase activity of tubulin. We find that this function is abolished by mutation in an arginine residue that is conserved in both cofactor C and RP2. Notably, mutations that alter this arginine codon cause familial retinitis pigmentosa. Our data imply that this residue acts as an "arginine finger" to trigger the tubulin GTPase activity and suggest that loss of this function in RP2 contributes to retinal degeneration. We also show that in Saccharomyces cerevisiae, both cofactor C and RP2 partially complement the microtubule phenotype resulting from deletion of the cofactor C homolog, demonstrating their functional overlap in vivo. Finally, we find that RP2 interacts with GTP-bound ADP ribosylation factor-like 3 protein, providing a link between RP2 and several retinal-specific proteins, mutations in which also cause retinitis pigmentosa.

Localization in the human retina of the X-linked retinitis pigmentosa protein RP2, its homologue cofactor C and the RP2 interacting protein Arl3.

Mutations in the retinitis pigmentosa 2 (RP2) gene cause a severe form of X-linked retinal degeneration. RP2 is a ubiquitous 350 amino acid plasma membrane-associated protein, which shares homology with the tubulin-specific chaperone cofactor C. RP2 protein, like cofactor C, stimulates the GTPase activity of tubulin in combination with cofactor D. RP2 has also been shown to interact with ADP ribosylation factor-like 3 (Arl3) in a nucleotide and myristoylation-dependant manner. In this study we have examined the relationship between RP2, cofactor C and Arl3 in patient-derived cell lines and in the retina. Examination of lymphoblastoid cells from patients with an Arg120stop nonsense mutation in RP2 revealed that the expression levels of cofactor C and Arl3 were not affected by the absence of RP2. In human retina, RP2 was localized to the plasma membrane of cells throughout the retina. RP2 was present at the plasma membrane in both rod and cone photoreceptors, extending from the outer segment through the inner segment to the synaptic terminals. There was no enrichment of RP2 staining in any photoreceptor organelle. In contrast, cofactor C and Arl3 localized predominantly to the photoreceptor connecting cilium in rod and cone photoreceptors. Cofactor C was cytoplasmic in distribution, whereas Arl3 localized to other microtubule structures within all cells. Arl3 behaved as a microtubule-associated protein: it co-localized with microtubules in HeLa cells and this was enhanced following microtubule stabilization with taxol. Furthermore, Arl3 co-purified with microtubules from bovine brain. Following microtubule depolymerization with nocodazole, Arl3 relocalized to the nuclear membrane. These data suggest that RP2 functions in concert with Arl3 to link the cell membrane with the cytoskeleton in photoreceptors as part of the cell signaling or vesicular transport machinery.