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...

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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.

Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14␣-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14␣-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of Candida albicans responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth. There was no evidence of cell leakage. Mass spectrometric analysis of the secretome showed that its composition was strongly affected and included 17 fluconazole-specific secretory proteins. Relative quantification of 14 N-labeled query walls relative to a reference standard mixture of 15 N-labeled yeast and hyphal walls in combination with immunological analysis revealed considerable fluconazole-induced changes in the wall proteome as well. They were, however, similar for both surface-grown and planktonic cultures. Two major trends emerged: (i) decreased incorporation of hypha-associated wall proteins (Als3, Hwp1, and Plb5), consistent with inhibition of hyphal growth, and (ii) increased incorporation of putative wall repair-related proteins (Crh11, Pga4, Phr1, Phr2, Pir1, and Sap9). As exposure to the wall-perturbing drug Congo red led to a similar response, these observations suggested that fluconazole affects the wall. In keeping with this, the resistance of fluconazole-treated cells to wall-perturbing compounds decreased. We propose that fluconazole affects the integrity of both the cellular membranes and the fungal wall and discuss its potential consequences for antifungal therapy. We also present candidate proteins from the secretome for clinical marker development.