The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum, but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores ϳ4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.Mycobacterium tuberculosis infects one-third of the world's population and kills 2 to 3 million people each year (13). The molecular and cellular mechanisms governing the pathogenesis of M. tuberculosis are beginning to be elucidated but are not fully understood. Mycobacterium marinum is a close relative of M. tuberculosis. M. marinum causes a tuberculosis-like disease in fish with symptoms similar to those of human tuberculosis and has been used as a surrogate model for studying the pathogenesis of M. tuberculosis (7,17,20,46,47).Previous studies have identified and partially characterized a specialized protein secretion system, ESX-1, in M. tuberculosis (14, 23, 24, 33, 44) and M. marinum (17, 50). This secretion system has recently been named the type VII secretion system (1). ESX-1 is encoded by genes of RD1 (region of difference 1) (24, 33, 44) and its surrounding region (23, 34), together termed extRD1 (4, 17). RD1 encompasses nine genes in M. tuberculosis (Rv3871 to Rv3879c) that are deleted from the attenuated vaccine Mycobacterium bovis BCG (2, 22). M. tuberculosis and M. marinum utilize ESX-1 to export virulence proteins that do not have the conventional SecA-dependent signal peptide sequences (17,24,33,44,50). The proteins that are secreted by ESX-1 and involved in virulence include ESAT-6, CFP-10, EspA, and Mh3881c (or EspB) (14,17,23,28,34,50). During secretion, Mh3881c is cleaved close to its C terminus to produce two fragments with apparent molecular masses of 50 and 11 kDa (28, 50). Inside the bacterial cytosol, the C-terminal sequence of Mh3881c interacts with ESA...

Ribosome synthesis involves dynamic association of ribosome biogenesis factors with evolving pre-ribosomal particles. Rio2 is an atypical protein kinase required for pre-40S subunit maturation. We report the crystal structure of eukaryotic Rio2 with bound ATP/Mg2+. Unexpectedly, the structure reveals a phosphoaspartate intermediate with ADP/Mg2+ in the active site, typically found in Na+-, K+- and Ca2+-ATPases. Consistent with this finding, ctRio2 exhibits a robust ATPase activity in vitro. In vivo, Rio2 docks on the ribosome with its active site occluded, and its flexible loop positioned to interact with the pre-40S subunit. Moreover, Rio2 catalytic activity is required for its dissociation from the ribosome, a necessary step in pre-40S maturation. We propose that phosphoryl transfer from ATP to Asp257 in Rio2’s active site and subsequent hydrolysis of the aspartylphosphate could be a trigger to power late cytoplasmic 40S subunit biogenesis.

The large family of Hox transcription factors has critical roles in early morphological development and later cell differentiation (McGinnis and Krumlauf 1992;Krumlauf 1994;Lawrence et al. 1996;Magli et al. 1997). Humans have 39 Hox genes arranged in four clusters (HoxAHoxD). The linear arrangement of genes within each cluster facilitates controlled spatial and temporal expression along the anterior-posterior axis of the body (Fig. 1A). Expression of Hox genes in the wrong segment at the wrong time often results in homeotic transformation of that segment (Lamka et al. 1992;Morgan et al. 1992;Duboule 1994a). Hox homeodomains contain identical DNA-base-contacting residues (Fig. 1B) and have very similar DNA sequence specificity, generally preferring a core DNA-binding site containing the sequence 5Ј-TTNAT-3Ј, whose third base depends on the particular Hox protein (Laughon 1991). Cooperative DNA binding with other protein cofactors, such as the PBC family of homeodomain proteins, is thought to enhance the specificity of Hox proteins and thereby allow them to carry out distinct functions (Chan et al. 1994;Chang et al. 1995;Mann and Chan 1996). The PBC family, which includes human Pbx1 and Drosophila Exd, belongs to a class of proteins that contains the TALE (three-aminoacid loop extension)-type homeodomain, so named because of a three-amino-acid insertion between homeodomain residues 23 and 24 in the loop between helices 1 and 2 (Mann 1995;Mann and Chan 1996;Bü rglin 1997). A subset of vertebrate Hox proteins interacts with Pbx1 via a conserved six-amino-acid motif, or hexapeptide, enabling cooperative DNA binding (Chang et al. 1995;Neuteboom et al. 1995;Peltenburg and Murre 1996;Shen et al. 1996Shen et al. , 1997Passner et al. 1999;Piper et al. 1999).Hox proteins required for anterior development are expressed earliest and are restricted to anterior domains. The more posterior proteins are expressed later in development and in more posterior domains (Duboule 1994b;Krumlauf 1994). This character is critical to their in vivo function. In general, when posterior proteins are ex-

The flavoprotein iodotyrosine deiodinase (IYD) salvages iodide from mono-and diiodotyrosine formed during the biosynthesis of the thyroid hormone thyroxine. Expression of a soluble domain of this membrane-bound enzyme provided sufficient material for crystallization and characterization by x-ray diffraction. The structures of IYD and two co-crystals containing substrates, mono-and diiodotyrosine, alternatively, were solved at resolutions of 2.0, 2.45, and 2.6 Å , respectively. The structure of IYD is homologous to others in the NADH oxidase/ flavin reductase superfamily, but the position of the active site lid in IYD defines a new subfamily within this group that includes BluB, an enzyme associated with vitamin B 12 biosynthesis. IYD and BluB also share key interactions involving their bound flavin mononucleotide that suggest a unique catalytic behavior within the superfamily. Substrate coordination to IYD induces formation of an additional helix and coil that act as an active site lid to shield the resulting substrate⅐flavin complex from solvent. This complex is stabilized by aromatic stacking and extensive hydrogen bonding between the substrate and flavin. The carbon-iodine bond of the substrate is positioned directly over the C-4a/N-5 region of the flavin to promote electron transfer. These structures now also provide a molecular basis for understanding thyroid disease based on mutations of IYD.The micronutrient iodide is essential for the biosynthesis of thyroxine (3,3Ј,5,5Ј-tetraiodothyronine), a hormone used by a wide range of organisms as a master control of metabolic rate. In mammals, iodide homeostasis in the thyroid gland is critical for generating thyroxine and is achieved by sequestering and salvaging iodide. Both of these functions are critical for human health, and congenital defects in either may lead to hypothyroidism (1, 2). Sequestration of iodide from the circulatory system is accomplished by a Na ϩ /I Ϫ symporter located in the plasma membrane of thyroid follicular cells (2). Salvage of iodide is accomplished by iodotyrosine deiodinase (IYD) 3 located in the apical plasma membrane surrounding the thyroid colloid in which thyroglobulin is stored and processed (3). Proteolysis of mature thyroglobulin releases thyroxine as well as mono-and diiodotyrosine (MIT and DIT, respectively). IYD catalyzes a reductive deiodination of MIT and DIT selectively to prevent loss of iodide that would otherwise occur by excretion of these amino acids. The gene encoding IYD has recently been identified (3, 4) and has provided an initial basis for correlating its mutation with hypothyroidism and goiter observed in certain patients (1). The crystal structure described in this work now supersedes the previous structural models.IYD represents one of only two enzymes known to promote reductive dehalogenation in mammals (Fig. 1). The other enzyme, iodothyronine deiodinase, acts alternatively to activate and deactivate thyroxine by deiodinating the outer or inner ring, respectively (5). Interestingly two distinct strateg...