{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/72352"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/72352","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Identification of the Matrix Targeting and Stop Transfer Domains in the Presequence of the Mitochondrial Intermembrane Space Protein Cytochrome C Peroxidase","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Kirchner, Sandra Chapman"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Kaput, James,"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014-12-17T21:58:39Z","date_published":"2014-12-17T21:58:39Z","updated_at":"2026-07-22T22:26:06Z","subjects":["Biology, Molecular"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(UMI)AAI9305582"],"render_values":[{"text":"(UMI)AAI9305582","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/72352","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Kaput, James,"]},{"key":"dc:creator","label":"Author","values":["Kirchner, Sandra Chapman"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2014-12-17T21:58:39Z","10000-01-01","1992"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology, Molecular"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/72352","(UMI)AAI9305582"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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.","Made available in DSpace on 2014-12-17T21:58:39Z (GMT). No. of bitstreams: 1 9305582.pdf: 5582447 bytes, checksum: af9fb956a64705eac3dd007d83ba9a03 (MD5) Previous issue date: 1992","Embargo set by: Seth Robbins for item 72520 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","148 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1992."]},{"key":"dc:title","label":"Title","values":["Identification of the Matrix Targeting and Stop Transfer Domains in the Presequence of the Mitochondrial Intermembrane Space Protein Cytochrome C Peroxidase"]}]}],"canonical_facts":{"dc:contributor":["Kaput, James,"],"dc:creator":["Kirchner, Sandra Chapman"],"dc:date":["2014-12-17T21:58:39Z","10000-01-01","1992"],"dc:description":["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.","Made available in DSpace on 2014-12-17T21:58:39Z (GMT). No. of bitstreams: 1 9305582.pdf: 5582447 bytes, checksum: af9fb956a64705eac3dd007d83ba9a03 (MD5) Previous issue date: 1992","Embargo set by: Seth Robbins for item 72520 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","148 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1992."],"dc:identifier":["http://hdl.handle.net/2142/72352","(UMI)AAI9305582"],"dc:subject":["Biology, Molecular"],"dc:title":["Identification of the Matrix Targeting and Stop Transfer Domains in the Presequence of the Mitochondrial Intermembrane Space Protein Cytochrome C Peroxidase"],"dc:type":["text"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:06Z"}