SUMO, a small ubiquitin-related modifier, is known to covalently attach to a number of nuclear regulatory proteins such as p53, I B, promyelocytic leukemia protein and c-Jun. The sumoylation reaction is catalyzed by the SUMO protease, which exposes the Cterminal active glycine residue of the nascent SUMO, the heterodimeric SUMO activating enzyme, the SUMO conjugating enzyme, Ubc9, and SUMO protein ligases, in a manner similar to ubiquitinylation. Identification of SUMO-regulated proteins is hampered by the fact that many sumoylated proteins are present at a level below normal detection limit. This limitation was overcome by either in vivo overexpression of Myc-SUMO or in vitro sumoylation with excess biotin-SUMO and Ubc9. Sumoylated proteins so obtained were affinity purified or isolated by immunoprecipitation. The isolated sumoylated proteins were identified by sequence analysis using mass spectrometric methods. Results reveal that several heterogeneous nuclear ribonucleoproteins (hnRNPs), zinc finger proteins, and nuclear pore complex proteins were sumoylated. The sumoylation of hnRNP A1, hnRNP F, and hnRNP K were confirmed in vivo by coimmunoprecipitation. In view of the facts that hnRNPs have been implicated in RNA splicing, transport, stability, and translation, our findings suggest that sumoylation could play an important role in regulating mRNA metabolism.R eversible covalent modification of proteins is a widely used regulatory mechanism for transmitting biological signals and for regulating the activity, biosynthesis, and degradation of major enzymes. This is due, in part, to its enormous capacity for integrating biological information and for signal amplification (1, 2). In addition to modification with low molecular weight modifiers, such as phosphorylation, nucleotidylation, and acetylation, ubiquitin is a well documented posttranslational protein modifier. Ubiquitinylation involves the covalent attachment of the C terminus of ubiquitin to the -amino moiety of a specific lysyl residue in modified proteins. In general, polyubiquitinylation tends to associate with the 26 S proteasome-mediated protein degradation, whereas monoubiquitinylation has been reported to be involved in receptor endocytosis, protein sorting, subnuclear trafficking, meiosis, and chromatin remodeling (3-5). To date, there are Ͼ10 known ubiquitin-like proteins that have been shown to ligate to other target protein molecules. Among them, small ubiquitin-related modifier (SUMO) is the most studied modifier. It possesses only an 18% identity in sequence homology to ubiquitin; nevertheless, its 3D structure is very similar to that of ubiquitin. SUMO has been shown to ligate to numerous proteins and modulate their translocation, activity, or stability (6-9). In view of the fact that, in mammalian cells, there are two more SUMO homologs, SUMO2 and SUMO3, additional functions of protein sumoylation have yet to be identified.Similar to ubiquitinylation, covalent attachment of SUMO to its target proteins requires three or four enzymes, namel...

Understanding the physiology and pathology of an organ composed of a variety of cell populations depends critically on genome-wide information on each cell type. Here, we report single-cell transcriptome profiling of over 6,800 freshly dispersed anterior pituitary cells from postpubertal male and female rats. Six pituitary-specific cell types were identified based on known marker genes and characterized: folliculostellate cells and hormone-producing corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Also identified were endothelial and blood cells from the pituitary capillary network. The expression of numerous developmental and neuroendocrine marker genes in both folliculostellate and hormone-producing cells supports that they have a common origin. For several genes, the validity of transcriptome analysis was confirmed by qRT-PCR and single cell immunocytochemistry. Folliculostellate cells exhibit impressive transcriptome diversity, indicating their major roles in production of endogenous ligands and detoxification enzymes, and organization of extracellular matrix. Transcriptome profiles of hormone-producing cells also indicate contributions toward those functions, while also clearly demonstrating their endocrine function. This survey highlights many novel genetic markers contributing to pituitary cell type identity, sexual dimorphism, and function, and points to relationships between hormone-producing and folliculostellate cells.

Three highly homologous small ubiquitin-related modifier (SUMO) proteins have been identified in mammals. Modifications of proteins by SUMO-1 have been shown to regulate transcription, nucleocytoplasmic transport, protein stability, and protein-protein interactions. Relative to SUMO-1, little is known about the functions of SUMO-2 or SUMO-3 (referred to as SUMO-2/3). Here, stable cell lines overexpressing processed forms of SUMO-2/3 (SUMO-2/3GG) as well as their non-conjugatable derivatives, SUMO-2/3⌬GG, were established. Cells overexpressing SUMO-2/3GG showed a premature senescence phenotype as revealed by cellular morphology and senescenceassociated galactosidase activity. The senescence pathway protein p21 was up-regulated in cells overexpressing SUMO-2/ 3GG. In contrast, cells overexpressing non-conjugatable forms of SUMO-2/3⌬GG showed neither an apparent senescent phenotype nor elevated p21. Both p53 and pRB were found to be modified by SUMO-2/3. Site-directed mutagenesis studies showed that Lys-386 of p53, the SUMO-1 modification site, is also the modification site for SUMO-2/3. In addition, H 2 O 2 treatment of untransfected cells caused an increase in p53 sumoylation by SUMO-2/3, whereas that by SUMO-1 remained unchanged. Moreover, knocking down tumor suppressor proteins p53 or pRB using small interfering RNA significantly alleviated the premature senescence phenotypes in SUMO-2/3GG overexpressing cells. Together, our results reveal that p53 and pRB can be sumoylated by SUMO-2/3 in vivo, and such modification of p53 and pRB may play roles in premature senescence and stress response.The amino acid sequence of the small ubiquitin-modifier (SUMO) 2 is only 18% identical to that of ubiquitin, but its threedimensional structure is very similar to ubiquitin. SUMO is attached to its target proteins by an enzymic mechanism that is analogous to the ubiquitinylation pathway (1-3). The mammalian SUMO family consists of SUMO-1, -2, and -3. Human SUMO-1 (SMT3C) exhibits 46% sequence identity with SUMO-2 (SMT3A) and SUMO-3 (SMT3B), whereas SUMO-2 and SUMO-3 are 96% identical in their processed forms; thus SUMO-2 and SUMO-3 are referred as SUMO-2/3 (4). SUMO-1 has been found to covalently modify Ͼ70 target proteins. Many of them are important regulatory proteins, such as p53, IB␣, c-Jun, promyelocytic leukemia protein (PML), and proliferating cell nuclear antigen (PCNA) (5, 6). The functions of SUMO-1 conjugation are target-specific and highly diverse. Sumoylation may be involved in the regulation of transcription, nucleocytoplasmic transport, DNA repair, protein stability, and chromosome separation (1, 2). Unlike SUMO-1, SUMO-2/3 can form polysumoylation chains due to their sequences containing the intrinsic SUMO consensus sequence KXE, where stands for a large hydrophobic amino acid residue (7). Another important difference between SUMO-2/3 and SUMO-1 conjugation pathways in mammalian cells is that the majority of SUMO-1 exists in the conjugated forms, whereas SUMO-2/3 exist primarily as free forms and readily conjug...