The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.

This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

The Wnt pathway controls numerous developmental processes via the ␤-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of ␤-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic ␤-catenin associates with APC and axin and is continuously phosphorylated by GSK-3␤, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3␤ from phosphorylating ␤-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of ␤-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces ␤-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate ␤-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3␤ and is both necessary and sufficient to initiate the ␤-catenin phosphorylation-degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway.

Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here we demonstrate that live influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes. Matrix protein 2 is sufficient and necessary for this inhibition of autophagosome degradation. Macroautophagy inhibition compromises cell survival of influenza virus infected cells, but does not influence viral replication. We propose that influenza A virus, which also encodes pro-apoptotic proteins, is able to determine the death of its host cell by inducing apoptosis and blocking macroautophagy.

We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 by SIRT3 activates the acetylCoA synthetase activity of AceCS2. This report identifies the first acetylated substrate protein of SIRT3. Our findings show that a mammalian sirtuin directly controls the activity of a metabolic enzyme by means of reversible lysine acetylation. Because the activity of a bacterial ortholog of AceCS2, called ACS, is controlled via deacetylation by a bacterial sirtuin protein, our observation highlights the conservation of a metabolic regulatory pathway from bacteria to humans.eversible lysine acetylation is a highly regulated posttranslational protein modification, which is controlled by protein deacetylases and acetyltransferases (1, 2). Although the importance of reversible lysine acetylation of nuclear nonhistone and histone proteins is well established, the role of protein modification by reversible lysine acetylation in mitochondria is unknown.The nicotinamide (NAM) adenine dinucleotide (NAD ϩ )-dependent deacetylase silent information regulator 2 (sir2) is an important mediator of longevity in response to caloric restriction signals in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster (3-9). Seven mammalian Sir2 homologs (SIRT1-7) are known (10-13). The recent discovery that SIRT3, SIRT4, and SIRT5 are found in mitochondria (14-17) suggests the existence of mitochondrial sirtuin substrate proteins. Results and DiscussionAs a strategy to identify lysine-acetylated mitochondrial proteins that could be targeted by SIRT3, -4, or -5, we searched for human proteins with sequence similarity to the region surrounding the acetylated lysine residue found in acetyl-CoA synthetase (ACS) from Salmonella enterica. We chose the acetyl-lysine-containing region of S. enterica ACS, because it is a known substrate for the sirtuin CobB (18,19). In a second step, human proteins showing high similarity were analyzed for their mitochondrial localization probability by using MITOPROT II and PREDOTAR Ver.