SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo. Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes. However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways. Here we use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast. These three approaches yielded overlapping sets of proteins with the most extensive set by far being those identified by mass spectrometry. The two-hybrid data also yielded a potential SUMO-binding motif. Functional categories of SUMO-modified proteins include SUMO conjugation system enzymes, chromatin-and gene silencingrelated factors, DNA repair and genome stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes. The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms.Many types of post-translational protein modifications alter protein function, and in some cases the modifying group is itself a protein. The prototypical example of this is ubiquitin, a small, highly conserved polypeptide that is reversibly linked to many different proteins (probably thousands) in the cell (1). Polypeptides distinct from but related to ubiquitin, called ubiquitinlike proteins or Ubls, can also be ligated to proteins (2, 3). Ligation to each Ubl has unique mechanistic and functional consequences. SUMO (Smt3 in the yeast Saccharomyces cerevisiae) is a divergent Ubl that has crucial roles in many organisms (4, 5). Vertebrates have four SUMO variants, SUMO1-SUMO4, whereas yeasts have only one. Only human SUMO1, and not the other SUMO variants, can substitute for the essential yeast protein (6).Activation and conjugation reactions involving SUMO have much in common with those of ubiquitin (reviewed in Refs. 2, 5, and 7). Attachment of substrates to Smt3/SUMO depends on a heterodimeric SUMO-activating enzyme (E1), 1 called Uba2-Aos1 in yeast, and a SUMO-conjugating enzyme (E2), Ubc9. Both enzymes form transient thiolester bonds with the C terminus of SUMO. Substrate recognition factors (E3s) that stimulate the transfer of SUMO from E2 to substrate have also been identified. SUMO, like ubiquitin, is always synthesized in precursor form, requiring enzymatic removal of a C-terminal peptide. Specialized proteases, called Ubl-specific proteases or Ulps, are responsible for these SUMO processing reactions and for reversing the post-translational attachment of SUMO to protein...

The nuclear pore complex (NPC) is both the major conduit for nucleocytoplasmic trafficking and a platform for organizing macromolecules at the nuclear envelope. We report that yeast Esc1, a non-NPC nuclear envelope protein, is required both for proper assembly of the nuclear basket, a structure extending into the nucleus from the NPC, and for normal NPC localization of the Ulp1 SUMO protease. In esc1Δ cells, Ulp1 and nuclear basket components Nup60 and Mlp1 no longer distribute broadly around the nuclear periphery, but co-localize in a small number of dense-staining perinuclear foci. Loss of Esc1 (or Nup60) alters SUMO conjugate accumulation and enhances ulp1 mutant defects. Similar to previous findings with Mlp1, both Esc1 and Ulp1 help retain unspliced pre-mRNAs in the nucleus. Therefore, these proteins are essential for proper nuclear basket function, which includes mRNA surveillance and regulation of SUMO protein dynamics. The results raise the possibility that NPC-localized protein desumoylation may be a key regulatory event preventing inappropriate pre-mRNA export.

The identity of the specific molecules required for the process of retinal circuitry formation is largely unknown. Here we report a newly identified zebrafish mutant in which the absence of the atypical cadherin, Celsr3, leads to a specific defect in the development of GABAergic signaling in the inner retina. This mutant lacks an optokinetic response (OKR), the ability to visually track rotating illuminated stripes, and develops a super-normal b-wave in the electroretinogram (ERG). We find that celsr3 mRNA is abundant in the amacrine and ganglion cells of the retina, however its loss does not affect synaptic lamination within the inner plexiform layer (IPL) or amacrine cell number. We localize the ERG defect pharmacologically to a late-stage disruption in GABAergic modulation of ON-bipolar cell pathway and find that the DNQX-sensitive fast b1 component of the ERG is specifically affected in this mutant. Consistently, we find an increase in GABA receptors on mutant ON-bipolar terminals, providing a direct link between the observed physiological changes and alterations in GABA signaling components. Finally, using blastula transplantation, we show that the lack of an OKR is due, at least partially, to Celsr3-mediated defects within the brain. These findings support the previously postulated inner retina origin for the b1 component and reveal a new role for Celsr3 in the normal development of ON visual pathway circuitry in the inner retina.

Synaptojanin 1 (SynJ1) is a polyphosphoinositide phosphatase involved in clathrin-mediated endocytosis in conventional synapses. Studies with the zebrafish mutant nrc have revealed that loss of SynJ1 also affects cone photoreceptor ribbon synapses, causing pronounced morphological and functional abnormalities. In this study we continue to examine the role of SynJ1 in photoreceptors. Using a newly generated antibody specific for zebrafish SynJ1, we localized this protein predominantly to cone photoreceptors. We then used blastula stage transplantation experiments to demonstrate that rods from nrc mutants lacking SynJ1 develop normally and do not have the pronounced morphological defects detected in cones. Given the known involvement of SynJ1 in synaptic vesicle endocytosis, we hypothesize that rods and cones use distinct mechanisms for vesicle recycling.

In many retinal diseases, the malfunction that results in photoreceptor loss occurs only in either rods or cones, but degeneration can progress from the affected cell type to its healthy neighbors. Specifically, in human and mouse models of Retinitis Pigmentosa the loss of rods results in the death of neighboring healthy cones. Significantly less is known about cone-initiated degenerations and their affect on neighboring cells. Sometimes rods remain normal after cone death, whereas other patients experience a loss of scotopic vision over time. The affect of cone death on neighboring cones is unknown. The zebrafish is a cone-rich animal model in which the potential for dying cones to kill neighboring healthy cones can be evaluated. We previously reported that the zebrafish cone phosphodiesterase mutant ( pde6c w59 ) displays a rapid death of cones soon after their formation and a subsequent loss of rods in the central retina. In this study we examine morphological changes associated with cone death in vivo in pde6c w59 fish. We then use blastulae transplantations to create chimeric fish with a photoreceptor layer of mixed wild-type (WT) and pde6c w59 cones. We find that the death of inoperative cones does not cause neighboring WT cone loss. The survival of WT cones is independent of transplant size and location within the retina. Furthermore, transplanted WT cones persist at least several weeks after the initial death of dysfunctional mutant cones. Our results suggest a potential for the therapeutic transplantation of healthy cones into an environment of damaged cones. IntroductionRods and cones are the primary light sensing cells of the visual system and form a mixed layer of cells in the outer retina. Cones are responsible for daytime and color vision whereas rods respond in low light levels. The phototransduction cascades of cones and rods are mechanistically analogous, but genetically distinct. Thus, mutations that abolish phototransduction in one cell type do not affect phototransduction in the other. However, in Retinitis Pigmentosa (RP), a rod-based disease, the death of inoperative rods causes the death of functional neighboring cones (Hartong et al., 2006). In patients, this secondary death manifests as tunnel vision and eventually blindness. The spread of death from damaged cells to their healthy neighbors is known as the bystander effect (Ripps, 2002). Understanding and controlling the bystander effect is one of the major challenges in the treatment of retinal degeneration.Retinal diseases are particularly complex due to the variability of the bystander effect between different types of photoreceptors. Studies in humans and mouse models have shown that rod degeneration frequently results in the death of neighboring cones, although the extent of cone degeneration can vary between patients (Ripps, 2002;Delyfer et al., 2004;Hartong et al., 2006). Mosaic mouse models have further shown that degenerating rods will kill neighboring functional rods (Huang et al., 1993;Kedzierski et al., 1998). Thus, rod degen...