Identification of the Matrix Targeting and Stop Transfer Domains in the Presequence of the Mitochondrial Intermembrane Space Protein Cytochrome C Peroxidase

Kirchner, Sandra Chapman

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Description

Title

Identification of the Matrix Targeting and Stop Transfer Domains in the Presequence of the Mitochondrial Intermembrane Space Protein Cytochrome C Peroxidase

Author(s)

Kirchner, Sandra Chapman

Issue Date

1992

Doctoral Committee Chair(s)

Kaput, James,

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

Biology, Molecular

Abstract

• The presequence of CCP, an intermembrane space heme protein, has been proposed to be composed of an amino-terminal basic domain, a stretch of hydrophobic residues and a basic, carboxy-terminal domain (Kaput et al, 1982). Results from previous in vitro import experiments with a mutant ccp that was missing ten alanines from its hydrophobic domain implied that this mutant was targeted to the mitochondrial matrix (Ekberg and Kaput, in preparation). This study utilized an in vivo approach to confirm the results obtained from analyzing import into isolated mitochondria and supporting the suggestion that the hydrophobic domain acts as a stop transfer sequence.
• Wild type ccp (CCP$\sb{\rm WT})$ and mutant ccp (CCP$\sb{\rm \Delta10A}$) presequences were synthesized using PCR and inserted at the 5$\sp\prime$ end of the gene for the mature $\Delta\sp1$-pyrolline-5-carboxylate dehydrogenase protein (PUT2). This protein is a component of the proline metabolic pathway and allows yeast cells to utilize proline as a sole nitrogen source. Yeast expression vectors were constructed from insertion of ars1, CEN3, URA3 and PUT upstream regulatory regions. When the vectors were transformed into a put- yeast strain, plated on ammonia media and transferred to proline medium, differences in growth patterns were observed. The CCP$\sb{\rm WT}$PUT strain did not grow while the CCP$\sb{\rm \Delta10A}$PUT strain did grow, albeit at a slower rate than a strain expressing prePUT. When the fusions were transcribed and translated in vitro and imported into isolated mitochondria, CCP$\sb{\rm \Delta10A}$PUT was targeted to a position behind the IM whereas CCP$\sb{\rm WT}$PUT was sensitive to proteases in both mitochondria and mitoplasts. In vivo localization experiments were also carried out and these confirmed the location of CCP$\sb{\rm \Delta 10A}$PUT behind the inner membrane. The sum of these results imply that the CCP$\sb{\rm WT}$ presequence is retained in the IM whereas a ccp signal missing part of its hydrophobic stretch traverses the bilayer. These results confirm the earlier observations of Ekberg and Kaput and are most compatible with the model that ccp is imported into the IMS via the Stop Transfer Pathway.

Graduation Semester

1992

Type of Resource

text

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