Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)‐anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide‐containing, mild base‐resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI‐anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI‐anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI‐anchored proteins.

To analyze the interaction of sorting signals with clathrin‐associated adaptor complexes, we developed an in vitro assay based on surface plasmon resonance analysis. This method monitors the binding of purified adaptors to immobilized oligopeptides in real time and determines binding kinetics and affinities. A peptide corresponding to the cytoplasmic domain of wild‐type influenza hemagglutinin, an apical membrane protein that is not endocytosed, did not significantly bind adaptor complexes. However, peptide sequences containing a tyrosine residue that has previously been shown to induce endocytosis and basolateral sorting were specifically recognized by adaptor complexes. The in vitro rates of adaptor association with these peptides correlated with the internalization rates of the corresponding hemagglutinin variants in vivo. Binding was observed both for purified AP‐2 adaptors of the plasma membrane and for AP‐1 adaptors of the Golgi, with similar apparent equilibrium dissociation constants in the range 10(‐7)‐10(‐6) M. Adaptor binding was also demonstrated for a sequence containing a C‐terminal di‐leucine sequence, the second major motif of endocytosis/basolateral sorting signals. These results confirm the concept that interaction of cytoplasmic signals with plasma membrane adaptors determines the endocytosis rate of membrane proteins, and suggest the model that clathrin‐coated vesicles of the trans‐Golgi network are involved in basolateral sorting.

Translocation and folding of proteins imported into mitochondria are mediated by two matrix‐localized chaperones, mhsp70 and hsp60. In order to investigate whether these chaperones act sequentially or in parallel, we studied their interaction with newly imported precursor proteins in isolated yeast mitochondria by coimmunoprecipitation. All precursors bound transiently to mhsp70. Release from mhsp70 required hydrolysis of ATP and did not immediately generate a tightly folded protein. For example, after imported mouse dihydrofolate reductase (a soluble monomeric enzyme) had been released from mhsp70, folding to a protease resistant conformation occurred only after a lag and was much slower than the release. Under standard import conditions, no significant association of DHFR with hsp60 could be detected. Similarly, newly imported hsp60 subunit was released from mhsp70 as an incompletely folded, unassembled intermediate which accumulated at low temperature and assembled to hsp60 14‐mer at higher temperature in an ATP‐dependent manner. Mas2p (the larger subunit of the MAS‐encoded processing protease) first bound to mhsp70, then to hsp60, and only then assembled with its partner subunit, Mas1p. We propose that ATP‐dependent release from mhsp70 is insufficient to cause folding of imported proteins and that assembly of hsp60 and Mas2p requires sequential, ATP‐dependent interactions with mhsp70 and hsp60.

Import of proteins into mitochondria involves the cooperation of protein translocation systems in the outer and inner membranes. We have identified a 45-k a protein at the protein import site of the yeast mitochondrial inner membrane. This 45-kDa protein could be crosslinked to a partly trocted precursor, which cannot be imported across the inner membrane when the matrix is depleted of ATP. In addition, an antibody against this protein strongly inhibited protein import into right-side-out inner-membrane vesicles. The 45-kDa protein accounts for only 0.1% of mitochondrial protei and appears peripherally attached to the outer face of the Inner membrane. The properties ofthis protein suggest that it is a component of the protein import system of the mitochondrial inner membrane.Import of proteins from the cytoplasm into the mitochondrial interior requires the concerted action of two distinct protein translocation systems, one in the outer membrane and the other in the inner membrane (1-3). The system in the outer membrane consists of several receptor proteins together with a set of transmembrane proteins that presumably form a protein transport channel across the outer membrane (1,4,5). One subunit of this putative outer-membrane channel was identified by crosslinking it to a precursor protein that was stuck across the protein import channels of both membranes (6). This import-site protein 42 (ISP42) proved an integral outer-membrane protein essential for viability (7). The Neurospora crassa homologue (termed MOM38) was shown to be part of a complex that also contained one of the import receptors (8).In contrast, no component of the inner-membrane translocation system has yet been identified. To Generation of Crosslinks. After translation, the Su9-DHFR-containing reticulocyte lysate was centrifuged for 20 mm in an Airfuge (Beckman) at 30 psi (1 psi = 6.9 kPa) and depleted of ATP by incubation for 10 min at 300C with apyrase (Sigma) at 50 units/ml. Mitochondria at 1 mg of protein per ml in import buffer (0.6 M sorbitol/50 mM Hepes-KOH, pH 7.4/25 mM KCI/10 mM MgCl2/2 mM KP1, pH 7.4/0.5 mM EDTA/fatty acid-depleted bovine serum albumin at 1 mg/ml) were depleted of ATP for 5 min at 300C in the presence of apyrase at 10 units/ml, oligomycin at 12.5 pug/ml, and efrapeptin at 1 ,ug/ml. The mitochondria were then reenergized by incubation with 2 mM NADH for 3 min at 300(. After 0.1 vol ofATP-depleted reticulocyte lysate was added, the mixture was incubated for 10 min at 300C and then put on ice. The membrane potential was collapsed by addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to 25 ,uM. Surface-bound precursor was degraded with trypsin (100 ,ug/ml, 20 min at 00C), followed by addition of soybean trypsin inhibitor to 1 mg/ml and phenylmethylsulfonyl fluoride to 1 mM. Mitochondria were reisolated by centrifugation (5 min at 15,000 x g) and resuspended in an equal volume of 0.6 M sorbitol/20 mM Hepes-KOH, pH 7.4. The crosslinking reaction was done by adding the cleavable, homobifunctional cross...

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross‐linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p‐beta‐galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right‐side‐out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.

A novel protein, belonging to the yeast family of FKBPs (FK-binding proteins), FKBP-70, was isolated from Saccharomyces cerevisiae by its interaction with the immunosuppressive agent FK-520. Its structural gene, FPR3, was cloned and the protein expressed and purified from Escherichia coli. This third member of the FKBP family in yeast is homologous to the other FKBPs at its carboxy terminus, showing conserved ligand binding and proline isomerase regions. It is, however, a longer acidic protein with several potential nuclear targeting sequences and a region of homology to nucleolins. Yeast strains deleted for FPR3, as well as a triple deletion mutant of this family of genes, FPRI, FPR2 and FPR3, are viable under normal conditions of growth, indicating that the FPR genes are not essential for life.