Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH2-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)–induced translation arrest and photo–cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH2-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter. Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation. 1) Three basic amino acid residues within the Cterminal five-residue segment (C-segment) contained the information required for mitochondrialtargeting. Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes. 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes. 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm. 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting. Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal. The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25.

Pressure-volume and pressure-radius relationships in lymphangions isolated from bovine mesenteric lymphatics were similar in pattern with those in the vein. Circumferential modulus of elasticity of the lymphatics ranged from 4.2 x 10(4) tatic walls. The contractile force increased in early stages of distension and decreased after an optimal intraluminal pressure was attained. The spontaneous activity was also affected by the rate of wall deformation. The pacemaker site of spontaneous activity seemed to be in the wall in the immediate vicinity of the inlet valve of a lymphangion. The activity propagated with a velocity of 4-5 mm/s. Ejection fraction of a lymphangion was between 45 and 65%. The endurance limit of the lymphatic valve was 68.4 +/- 7.6 cmH2O in specimens of about 3 mm in outer diameter. These findings suggested that lymphatic smooth muscle seemed to play a major role in elastic behavior of the wall and in regulation of the spontaneous activity, thereby affecting significantly passive and active lymph transport.

Co‐translational insertion of liver microsomal cytochrome P‐450 into the endoplasmic reticulum membrane is mediated by the signal recognition particle (SRP) and the presence in the cytochrome molecule of a signal sequence that can be recognized by SRP has been postulated. To locate this signal sequence, six hybrid cDNAs were constructed in which various segments of a cDNA for a rabbit liver cytochrome P‐450 are fused with a cDNA or its fragment encoding yeast porin (an outer mitochondrial membrane protein) or with a cDNA for pre‐interleukin 2 (a secretory protein) from which the 5′‐terminal portion encoding most of its signal sequence had been removed. These hybrid cDNAs were inserted into an SP‐6 transcription vector and transcribed in vitro. The mRNAs thus synthesized were translated in a cell‐free system in the presence of rough microsomes. It was thus found that only those chimeric proteins containing (at their amino‐terminal end) the amino‐terminal cytochrome P‐450 segments consisting of greater than or equal to 29 amino acid residues were co‐translationally inserted into the membrane in an SRP‐dependent fashion. These proteins were, however, neither processed nor translocated across the membrane. These findings, coupled with the observation that the major portion of these proteins, when inserted into the membrane, was degraded by trypsin, led to the conclusion that a short amino‐terminal segment (less than 29 residues) of the cytochrome P‐450 functions not only as an insertion signal but also as a stop‐transfer sequence. This segment is, therefore, similar to the internal signal of type II plasma membrane proteins, but differs from the latter in the topogenic function.