The C-terminal tail of tetraspanin protein CD9 contributes to its function and molecular organization.

Tetraspanin protein CD9 supports sperm-egg fusion, and regulates cell adhesion, motility, metastasis, proliferation and signaling. The large extracellular loop and transmembrane domains of CD9 engage in functionally important interactions with partner proteins. However, neither functional nor biochemical roles have been shown for the CD9 C-terminal tail, despite it being highly conserved throughout vertebrate species. To gain new insight into the CD9 tail, three C-terminal amino acids (Glu-Met-Val) were replaced with residues corresponding to C-terminal amino acids from tetraspanin protein CD82 (Pro-Lys-Tyr). Wild-type and mutant CD9 were then stably expressed in MOLT-4, K562, U937, RD and HT1080 cells. Whereas wild-type CD9 inhibited cell adhesion and spreading on fibronectin, mutant CD9 did not. Wild-type CD9 also promoted homotypic cell-cell aggregation and microvilli formation, whereas mutant CD9 did not. Protein interactions of wild-type and mutant CD9 were compared quantitatively using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with liquid-chromatography-tandem mass spectrometry (LC-MS/MS) technology. SILAC results showed that, despite wild-type and mutant CD9 having identical expression levels, mutant CD9 and its major transmembrane interacting partners were recovered in substantially reduced amounts from 1% Brij 96 lysates. Immunoprecipitation experiments confirmed that mutant CD9 recovery was decreased in Brij 96, but not in more stringent Triton X-100 detergent. Additionally, compared with wild-type CD9 complexes, mutant CD9 complexes were larger and more oligomerized in Brij 96 detergent, consistent with decreased Brij 96 solubility, perhaps due to more membrane domains packing more tightly together. In conclusion, multiple CD9 functions depend on its C-terminal tail, which affects the molecular organization of CD9 complexes, as manifested by their altered solubilization in Brij 96 and organization on the cell surface.

SUMO regulates the assembly and function of a cytoplasmic intermediate filament protein in C. elegans.

Sumoylation is a reversible posttranslational modification that plays roles in many processes, including transcriptional regulation, cell division, chromosome integrity, and DNA damage response. Using a proteomics approach, we identified approximately 250 candidate targets of sumoylation in C. elegans. One such target is the cytoplasmic intermediate filament (cIF) protein named IFB-1, which is expressed in hemidesmosome-like structures in the worm epidermis and is essential for embryonic elongation and maintenance of muscle attachment to the cuticle. In the absence of SUMO, IFB-1 formed ectopic filaments and protein aggregates in the lateral epidermis. Moreover, depletion of SUMO or mutation of the SUMO acceptor site on IFB-1 resulted in a reduction of its cytoplasmic soluble pool, leading to a decrease in its exchange rate within epidermal attachment structures. These observations indicate that SUMO regulates cIF assembly by maintaining a cytoplasmic pool of nonpolymerized IFB-1, and that this is necessary for normal IFB-1 function.

HP1 proteins form distinct complexes and mediate heterochromatic gene silencing by nonoverlapping mechanisms.

HP1 proteins are a highly conserved family of eukaryotic proteins that 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 nonoverlapping 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 nonoverlapping inhibitory mechanisms.

Ribosome binding of a single copy of the SecY complex: implications for protein translocation.

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.

Cdk1 coordinates cell-surface growth with the cell cycle.

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.

NMR analyses of the activation of the Arp2/3 complex by neuronal Wiskott-Aldrich syndrome protein.

The VCA domain of the neuronal Wiskott-Aldrich syndrome protein (N-WASP) is a potent activator of the Arp2/3 complex, a 240 kDa heteroheptameric actin-nucleating assembly. We used site-directed spin labeling of N-WASP peptides in conjunction with methyl-TROSY spectra of the intact, selectively labeled Arp2/3 complex to identify regions of the VCA that are proximal to the ARPC3 subunit of the assembly. We also cross-linked CA peptides to the Arp3, Arp2, ARPC1, and ARPC3 subunits. The combined data suggest that the extreme C-terminus of the A region and the C-terminus of the C region of N-WASP are proximal to ARPC3. These results have implications for the mechanism of Arp2/3 complex activation by VCA peptides. This study also demonstrates the utility of NMR spectroscopy for studying ligand binding events in large, asymmetric, macromolecular assemblies.

Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics.

Mass-spectrometry-based proteomics has become an essential tool for the qualitative and quantitative analysis of cellular systems. The biochemical complexity and functional diversity of the ubiquitin system are well suited to proteomic studies. This review summarizes advances involving the identification of ubiquitinated proteins, the elucidation of ubiquitin-modification sites and the determination of polyubiquitin chain linkages, as well as offering a perspective on the application of emerging technologies for mechanistic and functional studies of protein ubiquitination.

Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways.

IFN-alpha/beta plays an essential role in innate immunity against viral and bacterial infection. Among the proteins induced by IFN-alpha/beta are the ubiquitin-like ISG15 protein and its E1- (Ube1L) and E2- (UbcH8) conjugating enzymes, leading to the conjugation of ISG15 to cellular proteins. It is likely that ISG15 conjugation plays an important role in antiviral response because a human virus, influenza B virus, inhibits ISG15 conjugation. However, the biological function of ISG15 modification remains unknown, largely because only a few human ISG15 target proteins have been identified. Here we purify ISG15-modified proteins from IFN-beta-treated human (HeLa) cells by using double-affinity selection and use mass spectroscopy to identify a large number (158) of ISG15 target proteins. Eight of these proteins were subjected to further analysis and verified to be ISG15 modified in IFN-beta-treated cells, increasing the likelihood that most, if not all, targets identified by mass spectroscopy are bona fide ISG15 targets. Several of the targets are IFN-alpha/beta-induced antiviral proteins, including PKR, MxA, HuP56, and RIG-I, providing a rationale for the inhibition of ISG15 conjugation by influenza B virus. Most targets are constitutively expressed proteins that function in diverse cellular pathways, including RNA splicing, chromatin remodeling/polymerase II transcription, cytoskeleton organization and regulation, stress responses, and translation. These results indicate that ISG15 conjugation impacts nuclear as well as cytoplasmic functions. By targeting a wide array of constitutively expressed proteins, ISG15 conjugation greatly extends the repertoire of cellular functions that are affected by IFN-alpha/beta.

Proteomic insights into ubiquitin and ubiquitin-like proteins.

The dynamic and specific modification of cellular proteins by members of the ubiquitin protein family is a vital regulatory mechanism that lies at the heart of almost all biological processes. Because of both their pervasive and complex nature, these regulatory pathways have been the target of many recent proteomic studies. Such works have provided numerous insights. Through the use of various mass spectrometry techniques, affinity purification methods, and/or chemical probes, large lists have begun to be compiled for the multitude of substrates, interacting partners, and enzymatic components of these regulatory circuits. Furthermore, similar tools have provided many insights into functional aspects such as their mechanisms of substrate specificity and enzymatic activity. This review provides a summary of these recent proteomic works, along with comments on future directions of the field.

A proteomic strategy for gaining insights into protein sumoylation in yeast.

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.

Toward a general chemical method for rapidly mapping multi-protein complexes.

Ru(II)(bpy2)32+Cl2, ammonium persulfate, and visible light irradiation has been shown to rapidly and efficiently cross-link several interacting proteins. However, this methodology has not yet been used to map the architecture of large multi-protein complexes. In this study, this chemistry is applied to the crystallographically characterized yeast proteasome. The data obtained demonstrate both the method's increased generality and fidelity in comparison to traditional bifunctional cross-linking reagents, while also highlighting the future need for developing better analytical techniques to separate cross-linked products.

Using oxidative crosslinking and proximity labeling to quantitatively characterize protein-protein and protein-Peptide complexes.

The quantitative analysis of protein-protein and protein-peptide complexes is of fundamental importance in biochemistry. We report here that nickel-catalyzed proximity biotinylation and Ru(II)(bpy)(3)(2+)-mediated oxidative crosslinking can be used to measure the equilibrium dissociation constant and stoichiometry of protein complexes. Only small amounts of protein are required, neither of the binding partners must be immobilized on a surface, and no special instrumentation is necessary. This chemistry should provide a useful complement to existing methods for the analysis of protein-protein and protein-peptide interactions.