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PMID: 3025596 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae.

Molecular and cellular biology ·Vol. 6 ·No. 10 ·1986-10-00 ·Pages 3502-12

Brandriss MC, Krzywicki KA

Abstract

delta 1-Pyrroline-5-carboxylate (P5C) dehydrogenase, the second enzyme in the proline utilization (Put) pathway of Saccharomyces cerevisiae and the product of the PUT2 gene, was localized to the matrix compartment by a mitochondrial fractionation procedure. This result was confirmed by demonstrating that the enzyme had limited activity toward an externally added substrate that could not penetrate the inner mitochondrial membrane (latency). To learn more about the nature of the import of this enzyme, three gene fusions were constructed that carried 5'-regulatory sequences through codons 14, 124, or 366 of the PUT2 gene ligated to the lacZ gene of Escherichia coli. When these fusions were introduced into S. cerevisiae either on multicopy plasmids or stably integrated into the genome, proline-inducible beta-galactosidase was made. The shortest gene fusion, PUT2-lacZ14, caused the production of a high level of beta-galactosidase that was found exclusively in the cytoplasm. The PUT2-lacZ124 and PUT2-lacZ366 fusions made lower levels of beta-galactosidases that were mitochondrially localized. Mitochondrial fractionation and protease-protection experiments showed that the PUT2-lacZ124 hybrid protein was located exclusively in the matrix, while the PUT2-lacZ366 hybrid was found in the matrix as well as the inner membrane. Thus, the amino-terminal 124 amino acids of P5C dehydrogenase carries sufficient information to target and deliver beta-galactosidase to the matrix compartment. The expression of the longer hybrids had deleterious effects on cell growth; PUT2-lacZ366-containing strains failed to grow on proline as the sole source of nitrogen. In the presence of the longest hybrid beta-galactosidase, the wild-type P5C dehydrogenase was still properly localized in the matrix compartment, but its activity was reduced. The nature of the effects of these hybrid proteins on cell growth is discussed.

MeSH Terms
1-Pyrroline-5-Carboxylate Dehydrogenase DNA Restriction Enzymes Escherichia coli/genetics Galactosidases/genetics Genes Genes, Fungal Genotype Mitochondria/enzymology Nucleic Acid Hybridization Oxidoreductases Acting on CH-NH Group Donors/genetics Plasmids Saccharomyces cerevisiae/enzymology,genetics Saccharomyces cerevisiae Proteins beta-Galactosidase/genetics
Chemicals
Saccharomyces cerevisiae Proteins 1-Pyrroline-5-Carboxylate Dehydrogenase PUT2 protein, S cerevisiae Oxidoreductases Acting on CH-NH Group Donors DNA Restriction Enzymes Galactosidases beta-Galactosidase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Brandriss M C
Krzywicki K A
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.6.10.3502-3512.1986
Published
1986-10-00
Pages
3502-12
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC367099
Subset
IM
Grants
NIGMS NIH HHS · GM30405 · United States
External Links
PubMed source DOI: 10.1128/mcb.6.10.3502-3512.1986 PMC Full Text View journal details
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