Our interest in ISG15 originated in the course of experiments to elucidate the function of the NS1B protein of influenza B virus. We found that the NS1B protein binds ISG15 and inhibits its conjugation (6), indicating that ISG15 conjugation is likely to be an important part of the IFN-␣͞␤-induced antiviral response. However, it was not evident how ISG15 conjugation might serve such a role. To address this issue and to elucidate the function of ISG15 conjugation, we first identified the E1 and E2 enzymes in the ISG15 conjugation pathway as Ube1L and UbcH8, respectively, both of which are induced by IFN-␣͞␤ (6, 7). These findings enabled us to develop a system for a proteomics-based identification of ISG15 target proteins, which is described in the present study.We used this system to identify a large number (158) of ISG15 modified proteins in IFN-␤-treated human (HeLa) cells. The identity of these ISG15 target proteins provides insights into the function of ISG15 modification. Several of the targets are IFN-␣͞ ␤-induced antiviral proteins, providing a rationale for the inhibition of ISG15 conjugation by influenza B virus. Most targets are constitutively expressed human proteins that function in diverse cellular pathways, including RNA splicing, chromatin remodeling͞ polymerase II transcription, cytoskeleton organization and regulation, stress responses, and translation. These results indicate that ISG15 conjugation impacts nuclear as well as cytoplasmic functions and may have a role in regulating transcription and pre-mRNA splicing during the IFN-␣͞␤ response. Thus, by targeting this wide array of constitutively expressed proteins, ISG15 conjugation greatly extends the repertoire of cellular functions that are affected by IFN-␣͞␤. Materials and MethodsPlasmids. Plasmids containing the following PCR-generated reading frames were inserted into pcDNA3 vectors: Ube1L, UbcH8, His 6 -HA-ISG15, and His 6 -3xFLAG-ISG15. All of the cDNAs used for verifying ISG15 target proteins, except maspin, were generated by PCR by using a Human Leukocyte Matchmaker cDNA library (Clontech). The template for amplifying maspin was pEF-Maspin, provided by Zhang Min (Baylor School of Medicine, Houston). For the expression of V5-tagged target proteins, two modified pcDNA3 vectors containing the V5 epitope were constructed. The original BamHI site of pcDNA3 was eliminated and replaced by the V5 sequence followed by either a BamHI site (pcDNA3-V5-Bam) or a NotI site (pcDNA3-V5-Not). The PCR-generated reading frames for maspin, PTB-1, and thioredoxin reductase-1 (TrxR1) were cloned into pcDNA3-V5-Bam as BglII-BglII, BglII-R1, and BamH-R1 fragments, respectively. The PCR-generated reading frames for Hsp60 and moesin were cloned into pcDNA3-V5-Not as Not-XbaI and Not-EcoRI fragments, respectively. For the expression of 3xFLAG-RIG-I, its PCR-generated reading frame was inserted into the pCMV10 vector (Sigma).Purification of ISG15 Conjugates. HeLa cells in each of five 150-mm culture dishes (total of 10 8 cells) were transfected by using Fugene 6 ...

SUMMARY HP1 proteins are a highly conserved family of eukaryotic proteins, which bind to methylated histone H3 lysine 9 (H3K9) and are required for heterochromatic gene silencing. In fission yeast, two HP1 homologs, Swi6 and Chp2, function in heterochromatic gene silencing, but their relative contribution to silencing remains unknown. Here we show that Swi6 and Chp2 exist in non-overlapping complexes and make distinct contributions to silencing. Chp2 associates with the SHREC histone deacetylase complex (SHREC2), is required for histone H3 lysine 14 (H3K14) deacetylation, and mediates transcriptional repression by limiting RNA polymerase II access to heterochromatin. In contrast, Swi6 associates with a different set of nuclear proteins and with noncoding centromeric transcripts, and is required for efficient RNAi-dependent processing of these transcripts. Our findings reveal an unexpected role for Swi6 in RNAi-mediated gene silencing and suggest that different HP1 proteins ensure full heterochromatic gene silencing through largely non-overlapping inhibitory mechanisms.

Sumoylation represents a vital post-translational modification that pervades numerous aspects of cell biology, including protein targeting, transcriptional regulation, signal transduction, and cell division. However, despite its broad reaching effects, most biological outcomes of protein sumoylation remain poorly understood. In an effort to provide further insight into this complex process, a proteomics approach was undertaken to identify the targets of sumoylation en mass. Specifically, SUMO-conjugated proteins were isolated by a double-affinity purification procedure from a Saccharomyces cerevisiae strain engineered to express tagged SUMO. The components of the isolated protein mixture were then identified by subsequent LC-MS/MS analysis using an LTQ FT mass spectrometer. In this manner, 159 candidate sumoylated proteins were identified by two or more peptides. Furthermore, the high accuracy of the instrument, combined with stringent search criteria, enabled the identification of an additional 92 putative candidates by only one peptide. The validity of this proteomics approach was confirmed by performing subsequent Western blot experiments for numerous proteins and determining the actual sumoylation sites for several other substrates. These data combine with recent works to further our understanding of the breadth and impact of protein sumoylation in a diverse array of biological processes.

The mechanisms that control cell growth during the cell cycle are poorly understood. In budding yeast, cyclin dependent kinase 1 (Cdk1) triggers polarization of the actin cytoskeleton and bud emergence in late G1 through activation of the Cdc42 GTPase. However, Cdk1 is not thought to be required for subsequent growth of the bud. Here, we show that Cdk1 has an unexpected role in controlling bud growth after bud emergence. Moreover, we show that G1 cyclin-Cdk1 complexes specifically phosphorylate multiple proteins associated with Cdc24, the guanine nucleotide-exchange factor (GEF) that activates the Cdc42 GTPase. A mutant form of a Cdc24-associated protein that fails to undergo Cdk1-dependent phosphorylation causes defects in bud growth. These results provide a direct link between Cdk1 activity and the control of polarized cell growth.