Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme

The role of structural signals in intercompartmental transport has been addressed by the isolation of yeast invertase (SUC2) mutations that cause intracellular accumulation of active enzyme. Two mutations that delay transport of core-glycosylated invertase, but not acid phosphatase, have been mapped in the 5' coding region of SUC2. Both mutations reduce specifically the transport of invertase to a compartment, presumably in the Golgi body, where outer chain carbohydrate is added. Subsequent transport to the cell surface is not similarly delayed. One mutation (SUC2-s1) converts an ala codon to val at position -1 in the signal peptide; the other (SUC2-s2) changes a thr to an ile at position +64 in the mature protein. Mutation s1 results in about a 50-fold reduced rate of invertase transport to the Golgi body which is attributable to defective signal peptide cleavage. While peptide cleavage normally occurs at an ala-ser bond, the s1 mutant form is processed slowly at the adjacent ser-met position giving rise to mature invertase with an N-terminal met residue. s2 mutant invertase is transported about sevenfold more slowly than normal, with no delay in signal peptide cleavage, and no detectable abnormal physical property of the enzyme. This substitution may interfere with the interaction of invertase and a receptor that facilitates transport to the Golgi body

The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information

The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein

The Saccharomyces cerevisiae SEC14 Gene Encodes a Cytosolic Factor That Is Required for Transport of Secretory Proteins from the Yeast Golgi Complex

We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14ts defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species

The Amino Terminus of the Yeast F_1-ATPase β-Subunit Precursor Functions as a Mitochondrial Import Signal

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells

New component of ESCRT-I regulates endosomal sorting complex assembly

The endosomal sorting complex required for transport (ESCRT) complexes play a critical role in receptor down-regulation and retroviral budding. Although the crystal structures of two ESCRT complexes have been determined, the molecular mechanisms underlying the assembly and regulation of the ESCRT machinery are still poorly understood. We identify a new component of the ESCRT-I complex, multivesicular body sorting factor of 12 kD (Mvb12), and demonstrate that Mvb12 binds to the coiled-coil domain of the ESCRT-I subunit vacuolar protein sorting 23 (Vps23). We show that ESCRT-I adopts an oligomeric state in the cytosol, the formation of which requires the coiled-coil domain of Vps23, as well as Mvb12. Loss of Mvb12 results in the disassembly of the ESCRT-I oligomer and the formation of a stable complex of ESCRT-I and -II in the cytosol. We propose that Mvb12 stabilizes ESCRT-I in an oligomeric, inactive state in the cytosol to ensure that the ordered recruitment and assembly of ESCRT-I and -II is spatially and temporally restricted to the surface of the endosome after activation of the MVB sorting reaction