Most mitochondrial proteins are synthesized as preproteins on cytosolic polysomes and are subsequently imported into the organelle. The mitochondrial outer membrane contains a multisubunit preprotein translocase (Tom) which has receptors on the cytosolic side and a general import pore (GIP) in the membrane. Tom20-Tom22 and Tom70-Tom37 function as import receptors with a preference for preproteins that have amino-terminal presequences or internal targeting information, respectively. Tom40 is an essential constituent of the GIP, whereas Tom6 and Tom7 modulate the assembly and dissociation of the Tom machinery. Here we report the identification of Tom5, a small subunit that has a crucial role importing preproteins destined for all four mitochondrial subcompartments. Tom5 has a single membrane anchor and a cytosolic segment with a negative net charge, and accepts preproteins from the receptors and mediates their insertion into the GIP. We conclude that Tom5 represents a functional link between surface receptors and GIP, and is part of an 'acid chain' that guides the stepwise transport of positively charged mitochondrial targeting sequences.

The cytoplasmic region of the Ca2+-dependent cell-adhesion molecule (CAM) uvomorulin associates with distinct cytoplasmic proteins with molecular masses of 102, 88, and 80 kDa termed a, (3, and ycatenin, respectively. This complex formation links uvomorulin to the actin filament network, which seems to be of primary importance for its cell-adhesion properties. We show here that antibodies against a catenin also immunoprecipitate complexes that contain human N-cadherin, mouse P-cadherin, chicken A-CAM (adherens junction-specific CAM; also called N-cadherin) or Xenopus U-cadherin, demonstrating that a catenin is complexed with other cadherins. In immunofluoresence tests, a catenin is colocalized with cadherins at the plasma membrane. However, in cadherin-negative Ltk-cells, a catenin is found uniformly distributed in the cytoplasm, suggesting some additional biological function(s). Expression of uvomorulin in these cells results in a concentration of a catenin at membrane areas ofcell contacts. We also have cloned and sequenced murine a catenin.The deduced amino acid sequence reveals a signfMcant homology to vinculin. Our results suggest the possibility of a new vinculin-related protein family involved in the cytoplasmic anchorage of cell-cell and cell-substrate adhesion molecules.The cadherin gene family of Ca2l-dependent cell adhesion molecules (CAM) was originally composed of a rather limited number of transmembrane glycoproteins of which the best studied examples were uvomorulin/E-cadherin, liver CAM (L-CAM), N-cadherin, and P-cadherin (for a review, see refs. 1 and 2). Each member was found to regulate cell adhesion of particular cell types, and this was thought to be fundamental for the organization of multicellular organisms. More recently new members of this family have been described including M-cadherin on mouse myoblasts (3), E/P-, U-, and XB-cadherin in early Xenopus development (4-6), and a new subgroup of more distantly related desmosomal glycoproteins (7-9).It has been shown that the cytoplasmic region of uvomorulin associates with defined proteins of 102, 88, and 80 kDa termed a, f3, and y catenin, respectively (10). The MATERIALS AND METHODSCell Lines. Mouse fibroblasts Ltk-, human HeLa, chicken fibroblasts CEF38, and their respective transfectants expressing mouse uvomorulin, Li-i, H1-3, and C1-4 (10, 15) were used as well as embryonal carcinoma cells F9, PCC4, and PAS5E. Porcine kidney LLC-PK7 and Xenopus A6 cells were gifts from H. Hoschutzky (Freiburg, F.R.G.) and D. Wedlich (Berlin), respectively. The A6 cells were grown in Leibovitz L-15 medium containing 8% (vol/vol) fetal calf serum (FCS) at 240C. All other cells were cultured in Dulbecco's modified Eagle's medium containing 10%o FCS at 370C in an atmosphere containing 10%6 Co2. For the generation of F9 tumors, about 1 x 107 cells were injected subcutaneously in 129/SV mice, and solid tumors were removed 12-15 days later and stored at -80TC.Puriication of a Catenin. Ten grams of solid F9 tumor was homogenized in 50 ml of No...

The nucleotide sequence of several cDNA clones coding for the phosphate translocator from spinach chloroplasts has been determined. The cDNA clones were selected from a lambda gt10 library prepared from poly(A)+ mRNA of spinach leaves using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the isolated translocator protein. A 1439 bp insert of one of the clones codes for the entire 404 amino acid residues of the precursor protein corresponding to a mol. wt of 44,234. The full‐length clone includes 21 bp at the transcribed non‐coding 5′ region with the ribosome initiation sequence ACAATGG, a 1212 bp coding region and 199 bp at the non‐coding 3′ region excluding the poly(A) tail which starts 17 bp downstream from a putative polyadenylation signal, AATAAT. According to secondary structure predictions the mature part of the chloroplast phosphate translocator exhibits high hydrophobicity and consists of at least seven membrane‐spanning segments. Using plasmid‐programmed wheat germ lysate the precursor protein was synthesized in vitro and could be imported into spinach chloroplasts where it is inserted into the inner envelope membrane.

We have utilized a T7 polymerase/promoter system for the high‐level incorporation of methionine analogs with suitable labels for structural research (X‐ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high‐level substitution of methionine by 2‐aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X‐ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high‐level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine‐limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high‐level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy‐atom derivatives suitable for X‐ray structural analysis, supplementing the traditional trial‐and‐error preparation of heavy‐atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high‐level incorporation of amino acid analogs can provide single‐atom mutations for the detailed study of the structure and function of proteins.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of proteins, electroblotted onto polymer membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation is demonstrated. The proteins are desorbed directly from the blot membranes after matrix application. Desorption with 2.94-^m infrared radiation and succinic acid as matrix was found superior to 355-nm UV desorption using 2,5-dihydroxybenzoic acid as matrix. Several commercially available membranes tested resulted in protein signals after matrix incubation of the membrane. Systematic