Christoph Eckerskorn

Tom5 functionally links mitochondrial preprotein receptors to the general import pore

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.

Existence of a molecular ruler in proteasomes suggested by analysis of degradation products

Analysis of the degradation products from two proteins, the insulin B‐chain and human hemoglobin, generated by archaebacterial Thermoplasma acidophilum 20 S proteasomes, revealed an unexpectedly broad specificity. In spite of the vast number of different peptides found, they fell into a rather narrow size range. This suggests that a molecular ruler exists which determines the length of the cleavage products.

The fusicoccin receptor of plants is a member of the 14-3-3 superfamily of eukaryotic regulatory proteins

The receptor for the wilt‐inducing phytotoxin fusicoccin was purified to homogeneity from plasma membranes of Commelina communis as a complex with the radioligand [3H]9′‐nor‐8′‐hydroxyfusicoccin. The preparation consisted of two polypeptides with apparent molecular masses of 30.5 kDa and 31.5 kDa and with isoelectric points of around pH 5.2 and 5.3, respectively. The proteins were N‐terminally blocked. Internal amino acid sequences were obtained for both polypeptides of the fusicoccin‐binding complex. Sequence information, as well as subsequent immunological analysis, proved that both polypeptides are members of the eukaryotic 14‐3‐3 family, which comprises structurally conserved regulatory proteins of widespread occurrence and a wide range of functions. 14‐3‐3 isoform(s) constituting the fusicoccin receptor are distinguishable from other cellular 14‐3‐3 proteins by their tight association with the plasma membrane. Applying temperature‐induced Triton X‐114 phase separation experiments, they, as well as the target enzyme of fusicoccin action, the H+‐ATPase, partitioned into the phospholipid‐rich fraction which contains the most hydrophobic proteins. The results discussed herein provide a basis for the elucidation of the molecular mechanism of fusicoccin action.

Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema

A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.

Molecular cloning, sequence analysis and elicitor-/ozone-induced accumulation of cinnamyl alcohol dehydrogenase from Norway spruce (Picea abies L.)

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme from Norway spruce (Picea abies L.) cell culture. Here we report on partial protein sequences of the 42 kDa CAD polypeptide. A cDNA encoding CAD was isolated from the spruce cell culture. The open reading frame of a full-length cDNA coded for a 357 amino acid polypeptide with a calculated Mr of 38 777 Da. The identity of the deduced polypeptide was verified by comparison with amino acid sequences of tryptic peptides from the purified enzyme. Southern blot analysis showed the presence of only one gene for CAD. Sequence comparison with CAD from tobacco and with a N-terminal protein sequence from loblolly pine CAD showed an identity of 69.7% and 91.5%, respectively. Treatment of spruce cell cultures with elicitor, as well as of seedlings with ozone both markedly increased the CAD mRNA level.

Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins

The human plasma protein patterns obtained by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) is a good model system for post‐translational modifications because of the existence of several „ladders”︁ of protein spots [Anderson, N. L., Anderson, N. G., Electrophoresis 1991, 12, 883—906], so‐called „trains”︁ of spots. Our investigation of several proteins, amongst others beta2‐microglobulin and the haptoglobin chains, found the differences in isoelectric points (pI) to be due to deamidation of asparagines. After enzymatic cleavage with endopeptidases in the 2‐D polyacrylamide gel, the asparagine and deamidated asparagine containing peptides were separated and quantified by reversed‐phase HPLC. In order to separate these peptides, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine‐containing and deamidated asparagine‐containing peptides increased. But how do deamidated asparagines contribute to the observed spot pattern? One spot in the 2‐D gel consists of a mixture of protein species with the same number of deamidated asparagines but on different sequence position sites. The difference between the spots in the „ladder”︁ is a growing number of negative charges introduced in the protein by an increasing number of deamidated asparagines. As a consequence, the mass difference between two spots is exactly 1 Da, which is shown in this paper for intact protein masses and the corresponding deamidated peptides.

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-l-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20–56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

SKAP-HOM, a novel adaptor protein homologous to the FYN-associated protein SKAP551

A recombinant GST‐Fyn‐SH2 domain was used to purify proteins from lysates of pervanadate treated EL4 cells. N‐terminal sequencing and molecular cloning of one of the purified polypeptides resulted in the identification of a novel adaptor protein that shares strong structural homology to the recently cloned Fyn‐associated adaptor protein SKAP55. This protein was termed SKAP‐HOM ( 55 ologue). Despite their striking homology, SKAP55 and SKAP‐HOM have distinct characteristics. Thus, unlike SKAP55, which is exclusively expressed in T lymphocytes, SKAP‐HOM expression is ubiquitous. Furthermore, while SKAP55 is constitutively tyrosine phosphorylated in resting human T cells, SKAP‐HOM is expressed as a non‐phosphorylated protein in the absence of external stimulus but becomes phosphorylated following T cell activation. In addition, SKAP‐HOM does not associate with p59fyn in T cells although it represents a specific substrate for the kinase in COS cells. Finally, we demonstrate that, as previously shown for SKAP55, SKAP‐HOM interacts with the recently identified polypeptide SLAP‐130.

Sequence analysis of proteolytic fragments of 124-kilodalton phytochrome from etiolatedAvena sativa L.: Conclusions on the conformation of the native protein.

Proteolytic fragments were obtained by limited proteolysis of 124-kDa (kilodalton) phytochrome from etiolatedAvena sativa using trypsin, endoproteinase-Lys-C, endoproteinase-Glu-C and subtilisin. The fragments were separated by sodium dodecyl sulfate gel electrophoresis, blotted onto activated glass-fiber sheets and investigated by amino-acid sequencing in a gas-phase sequencer. Determination of N-terminal sequences in three to six Edman degradation steps allowed the exact localization of the fragments within the published entire amino-acid sequence of 124-kDaAvena phytochrome (H.P. Hershey, R.F. Barker, K.B. Idler, J.L. Lissemore, P.H. Quail (1985), Nucleic Acids Res.13, 8543–8559). From the knowledge of the exact sites for preferred proteolytic cleavage of undenatured phytochrome, conclusions on the conformation of the phytochrome protein were drawn. Sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are considered to be localized in the interior of the native phytochrome. Two different sites which are exposed in the far-red-absorbing form but not in the red-absorbing form of phytochrome are localized at amino-acid residues 354 and 753, respectively. The N-terminal region which is exposed only in the red-absorbing form stretches only as far as amino-acid residue 60.

Proteasome function is dispensable under normal but not under heat shock conditions in thermoplasma acidophilum

Hitherto the biology of proteolysis in prokaryotes, particularly in archaea, is only poorly understood. We have used the tri‐peptide vinyl sulfone inhibitor carboxybenzyl‐leucyl‐leucyl‐leucine vinyl sulfone (Z‐L3VS) to study the in vivo function of proteasomes in Thermoplasma acidophilum. Z‐L3VS is a potent inhibitor of the Thermoplasma proteasome and is capable of modifying 75 to 80% of the proteasomal β‐subunits in cell cultures. Inhibition of proteasomes has only marginal effects under normal growth conditions. Under heat shock conditions, however, the effects of proteasome inhibition are much more severe, to the extent of complete cell growth arrest. These data suggest that other proteolytic systems may exist that can compensate for the loss of proteasome function in T. acidophilum.