We have probed the environment of a precursor protein stuck in mitochondrial import sites using cleavable bifunctional crosslinking reagents. The stuck precursor was crosslinked to a 70 kd protein which, by immunological techniques, was shown to be a matrix protein. The protein was purified to homogeneity by ATP‐Sepharose chromatography and partially sequenced. Fourteen of its 15 N‐terminal amino acids were identical to residues 24–38 of the protein encoded by the nuclear gene SSC1, which had been proposed to encode a dnaK‐like 70 kd mitochondrial stress protein. Our data imply that this mitochondrial hsp70 is made with a cleavable matrix‐targeting sequence composed of 23 residues. The complex containing stuck precursor, mitochondrial hsp70, and ISP42 could be solubilized from mitochondria by the non‐ionic detergent Triton X‐100 even without crosslinking, suggesting tight association of these three components. As the stuck precursor is arrested at an early stage of translocation, mitochondrial hsp70 may initiate the events that lead to refolding of imported precursors in the matrix space.

A purified mitochondrial precursor protein unfolds to a protease‐sensitive conformation at the surface of isolated mitochondria before being imported into the organelles. This unfolding is stimulated by a potential across the mitochondrial inner membrane, but does not require ATP. In contrast, import of the surface‐bound unfolded precursor requires ATP, but no potential; it is accompanied by a refolding inside the mitochondria.

SUlTllnaryNaive T cells are selectively recruited from the blood into peripheral lymph nodes during lymphocyte recirculation. L-selectin, a lectin-like receptor, mediates the initial attachment oflymphocytes to high endothelial venules (HEV) in lymph nodes. A subsequent step involving the activation of {32 integrins has been proposed to facilitate firm adhesion, but the activating signals are poorly understood. We report here that either antibody-mediated cross-linking of L-selectin on human lymphocytes or treatment of the ceils with GlyCAM-1, an HEV-derived, secreted ligand for L-selectin, stimulates their binding to ICAM-1 through the {32 integrin pathway. Furthermore, GlyCAM-1 causes the rapid expression ofa neoepitope on {32 integrins associated with a high-avidity state. Naive (CD45RA+), but not memory (CD45P,.0 +) lymphocytes, respond to L-selectin cross-linking or GlyCAM-1 treatment. Thus, the complexing of L-selectin by specific ligands may provide key signals to naive lymphocytes, contributing to their selective recruitment into peripheral lymphoid organs.

Abstract. Import of precursor proteins into the yeast mitochondrial matrix can occur directly across the inner membrane. First, disruption of the outer membrane restores protein import to mitochondria whose normal import sites have been blocked by an antibody against the outer membrane or by a chimeric, incompletely translocated precursor protein. Second, a potential-and ATP-dependent import of authentic or artificial precursor proteins is observed with purified inner membrane vesicles virtually free of outer membrane components. Third, import into purified inner membrane vesicles is insensitive to antibody against the outer membrane. Thus, while outer membrane components are clearly required in vivo, the inner membrane contains a complete protein translocation system that can operate by itself if the outer membrane barrier is removed.

No abstract