Abstract. Bcl-2 is a mitochondrial-and perinuclearassociated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2c~ for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2ot. A truncation mutant of bcl-2ot lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-o~-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-I/) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg -Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As braininfiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system.

SlllllmaryCytolytic T lymphocytes (CTL), natural killer cells, and lymphokine-activated killer (LAK) cells are cytolytic cells known to release the cytolytic protein perforin and a family of proteases, named granzymes, from cytoplasmic stores upon interaction with target cells. We now report the purification of an additional major 60-kD granule-associated protein (grp 60) from human LAK cells and from mouse cytolytic T cells. The NH2-terminal amino acid sequence of the polypeptide was found to be identical to calreticulin. Calreticulin is a calcium storage protein and carries a COOH-terminal KDEL sequence, known to act as a retention signal for proteins destined to the lumen of the endoplasmic reticulum. In CTLs, however, calreticulin colocalizes with the lytic perforin to the lysosome-like secretory granules, as confirmed by double label immunofluorescence confocal microscopy. Moreover, when the release of granule-assodated proteins was triggered by stimulation of the T cell receptor complex, calreticulin was released along with granzymes A and D. Since perforin is activated and becomes lyric in the presence of calcium, we propose that the role of calreticulin is to prevent organelle autolysis due to the protein's calcium chelator capacity.

Abstract. Transepithelial Na ÷ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosteroneinduced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K ÷-ATPase was mainly mediated by a two-to fourfold accumulation of both a-and 13-subunit mRNAs. The specific competitor spironolactone decreased basal Na + transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosteronedependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.

Polyclonal antibodies raised against the alpha- and beta-subunits of amphibian kidney Na+-K+-ATPase were used to screen an expression library from Xenopus laevis kidney epithelial cells (A6 cell line). cDNAs coding for each Na+-K+-ATPase subunit were identified and used to isolate near full-length cDNAs. The complete nucleotide sequence and the deduced amino acid sequence were determined. The alpha-subunit is an alpha (alpha I)-isoform. The alpha- and beta-subunits are more closely related to the mammalian and avian than the fish sequences. Antibodies raised against the fusion proteins produced by the two clones served to immunoprecipitate proteins from biosynthetically labeled or selectively surface-radioiodinated A6 cells grown on a porous substrate. The alpha- and the beta-subunits of Na+-K+-ATPase were found associated early in the course of biosynthesis and were restricted to the basolateral plasma membrane.