Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co‐overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse–chase experiments with mitochondria isolated from phb1/phb2‐null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins.

Abstract. Several nuclear activities and components are concentrated in discrete nuclear compartments. To understand the functional significance of nuclear compartmentalization, knowledge on the spatial distribution of transcriptionally active chromatin is essential. We have examined the distribution of sites of transcription by RNA polymerase II (RPII) by labeling nascent RNA with 5-bromouridine 5'-triphosphate, in vitro and in vivo. Nascent RPII transcripts were found in over 100 defined areas, scattered throughout the nucleoplasm. No preferential localization was observed in either the nuclear interior or the periphery. Each transcription site may represent the activity of a single gene or, considering the number of active pre-mRNA genes in a cell, of a cluster of active genes. The relation between the distribution of nascent RPII transcripts and that of the essential splicing factor SC-35 was investigated in double labeling experiments. Antibodies against SC-35 recognize a number of welldefined, intensely labeled nuclear domains, in addition to labeling of more diffuse areas between these domains (Spector, D. L., X. -D. Fu, and T. Maniatis.1991. EMBO (Eur. Mol. Biol. Organ.)J. 10:3467-3481). We observe no correlation between intensely labeled SC-35 domains and sites of pre-mRNA synthesis. However, many sites of RPII synthesis colocalize with weakly stained areas. This implies that cotranscriptional splicing takes place in these weakly stained areas. These areas may also be sites where splicing is completed posttranscriptionally. Intensely labeled SC-35 domains may function as sites for assembly, storage, or regeneration of splicing components, or as compartments for degradation of introns.T HE cell nucleus comprises all factors required for faithful replication of the genome and regulated synthesis, processing and transport of RNA. In recent years much information on nuclear organization has become available. It is clear now that the nucleus is highly organized (reviewed by Jackson, 1991; van Driel et al., 1991). The most conspicuous subnuclear domain is the nucleolus in which ribosomal genes from different chromosomes are clustered and ribosome assembly takes place (reviewed by Scheer and Benavente, 1990; HernandezVerdun, 1991). Other examples of a domainlike organization in the nucleus are: replication clusters during S-phase (reviewed by Berezney, 1991), clustered splicing components (Spector, 1990;Fu and Maniatis, 1990; CarmoFonseca et al., 1992), hnRNP proteins (Pifiol-Roma et al., 1989; Ghetti et al., 1992), and tracks and foci of specific RNAs (Lawrence and Singer, 1991; Huang and Spector, 1991). In addition, a number of structures have been visualized of which the function is still unknown (Ascoli and Maul, 1991;Saunders et al., 1991; Ra~ka et al., 1991; al., 1992). The structural basis of the occurrence of nuclear activities in domains and the functional significance of this organizing principle for the regulation of gene expression and DNA replication are not understood.Essential for understandi...

Nuclear bodies (NBs) are ultrastructurally defined granules predominantly found in dividing cells. Here we show that PML, a protein involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), is specifically bound to a NB. PML and several NB‐associated proteins, found as auto‐antigens in primary biliary cirrhosis (PBC), are co‐localized and co‐regulated. The APL‐derived PML‐RAR alpha fusion protein is shown to be predominantly localized in the cytoplasm, whereas a fraction is nuclear and delocalizes the NB antigens to multiple smaller nuclear clusters devoid of ultrastructural organization. RA administration (which in APL patients induces blast differentiation and consequently complete remissions) causes the re‐aggregation of PML and PBC auto‐antigens onto the NB, while PML‐RAR alpha remains mainly cytoplasmic. Thus, PML‐RAR alpha expression leads to a RA‐reversible alteration of a nuclear domain. These results shed a new light on the pathogenesis of APL and provide a molecular link between NBs and oncogenesis.

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The mitochondrial prohibitin complex consists of two subunits (PHB1 of 32 kD and PHB2 of 34 kD), assembled into a membrane-associated supercomplex of approximately 1 MD. A chaperone-like function in holding and assembling newly synthesized mitochondrial polypeptide chains has been proposed. To further elucidate the function of this complex, structural information is necessary. In this study we use chemical crosslinking, connecting lysine side chains, which are well scattered along the sequence. Crosslinked peptides from protease digested prohibitin complexes were identified with mass spectrometry. From these results, spatial restraints for possible protein conformation were obtained. Many interaction sites between PHB1 and PHB2 were found, whereas no homodimeric interactions were observed. Secondary and tertiary structural predictions were made using several algorithms and the models best fitting the spatial restraints were selected for further evaluation. From the structure predictions and the crosslink data we derived a structural building block of one PHB1 and one PHB2 subunit, strongly intertwined along most of their length. The size of the complex implies that approximately 14 of these building blocks are present. Each unit contains a putative transmembrane helix in PHB2. Taken together with the unit building block we postulate a circular palisade-like arrangement of the building blocks projecting into the intermembrane space.Keywords: Crosslinking; mass spectrometry; prohibitin complex; PHB complex; structure predictionThe two structurally related proteins PHB1 and PHB2, previously referred to as prohibitin proteins, localize to mitochondria in mammals, plants, and yeast (Ikonen et al. 1995;Coates et al. 1997;Snedden and Fromm 1997;Berger and Yaffe 1998;Steglich et al. 1999;Nijtmans et al. 2002). In yeast, levels of PHB1 and PHB2 have shown to be interdependent, and have been shown to physically associate with each other to form a large multimeric complex in the mitochondrial inner membrane of mammals and yeast (Snedden and Fromm 1997;Steglich et al. 1999;Nijtmans et al. 2000). The molecular mass of the so-called PHB complex is estimated to be 1 MD by migration in Blue Native Electrophoresis (BNE) experiments. PHB constituent proteins are ubiquitously expressed in mammalian tissues, and have been highly conserved through evolution, suggesting a vital function among eukaryotes. To date, various functions have been attributed to both PHB proteins, including cell cycle Reprint requests to: Jaap Willem Back, SILS/Mass Spectrometry Group, Nieuwe Achtergracht 166, 1018WV Amsterdam, The Netherlands; e-mail: jwback@science.uva.nl; fax: 31(20)5256568.Abbreviations: PHB, prohibitin; DTSP, dithiobis(succinimidylpropionate); sBID,sulfo-N-benzyliminodiacetoylhydroxysuccinimid; BNE, blue native gel electrophoresis; MALDI, matrix-assisted laser desorption ionization; ESI, electrospray ionization; TOF, time of flight; MS, mass spectrometry; MSMS, low energy collision experiments; CID, collision-induced dissociatio...

A new method is presented to screen proteolytic mass maps of cross-linked protein complexes for the presence of cross-linked peptides and for the verification of proposed structures. On the basis of the incorporation of 18O from isotopically enriched water into the C-termini of proteolytic peptides, cross-linked peptides are readily distinguished in mass spectra by a characteristic 8 amu shift. This is due to the incorporation of two 18O atoms in each C-terminus, so that normal and surface-labeled peptides shift 4 amu and cross-linked peptides containing two C-termini will shift 8 amu compared with their unlabeled counterparts. The method is fast, sensitive, and reliable and can be combined with any available cross-linking reagent and a wide range of proteolytic agents. As proof of principle, we successfully applied the method to a complex of two DNA repair proteins (Rad18-Rad6) and identified the interaction domain.