Like its mitochondrial homolog Oxa1p, the inner membrane protein YidC of Escherichia coli is involved in the integration of membrane proteins. We have analyzed individual insertion steps of the polytopic E. coli membrane protein MtlA targeted as ribosome-nascent chain complexes to inner membrane vesicles. YidC can accommodate at least the first two transmembrane segments of MtlA at the protein lipid interface and retain them even though the length of the nascent chain would amply allow insertion into membrane lipids. An even longer insertion intermediate of MtlA is described that still has the first transmembrane helix bound to YidC while the third contacts SecE and YidC during integration. Our findings suggest that YidC forms a contiguous integration unit with the SecYE translocon and functions as an assembly site for polytopic membrane proteins mediating the formation of helix bundles prior to their release into the membrane lipids.

The twin-arginine translocation (Tat) pathway exports those precursor proteins to the periplasmic space of bacteria that harbor a twin-arginine (RR) consensus motif in their signal sequences. We have reproduced translocation of several Tat substrates into inside-out plasma membrane vesicles from Escherichia coli. Translocation proceeding at an efficiency of up to 20% occurs specifically via the Tat pathway as indicated by (i) its requirement for elevated levels of the TatABC proteins in the membrane vesicles, (ii) competition by an intact twin-arginine signal peptide, and (iii) susceptibility toward dissipation of the transmembrane H ؉ gradient. The latter treatment, while blocking translocation, still allows for functional membrane association of Tat precursors. This is shown by the finding that translocation of isolated membrane-bound Tat precursor is restored upon re-energization of the vesicles.

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The highmolecular-mass band occurred in low quantity and consisted of, probably eight, apparently identical chains of M , = 33 000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with MF = 63000-65000 and M! = 61000-62000. The N termini of both chains were threonine, whereas their C-terminal sequences were different:Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different.In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess ( c I~)~ structure, the apoenzyme a/?. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase.Pyruvate decarboxylase (PDC) is known to occur in yeasts, several other fungi, many plant materials (review: [l]) and a few bacteria [2, 31. One of the early sources for its preparation has been wheat germ [4]. When our efforts to achieve a detailed chemical analysis of yeast PDC became hampered by uncontrollable actions of contaminating proteinases [5], a practical alternative seemed to be the investigation of PDC from wheat germ, which was expected to contain fewer proteinases. The original method for the preparation of wheat germ PDC [4] has been applied without major alterations in all instances when this enzyme was hitherto needed for a variety of purposes [6 -111. Re-examination of this procedure with modern electrophoresis methods demonstrated that it yields PDC of insufficient chain homogeneity for the requirements of peptide mapping and sequence analysis. Therefore a more efficient isolation scheme was elaborated.

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