Import of proteins along the presequence pathway

Import of proteins along the presequence pathway

Alexander Benjamin Schendzielorz

Doctoral thesis

Date of Examination: 2017-11-15

Date of issue: 2017-12-01

Advisor: Rehling, Peter Prof. Dr.

Referee: Rehling, Peter Prof. Dr.

Referee: Rodnina, Marina Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0023-3F95-8

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Abstract

English

The aim of this study was to investigate the mechanism of protein transport by the presequence translocase (TIM23) in the inner mitochondrial membrane. Presequencecontaining proteins are imported through the translocase of the outer membrane (TOM) complex and handed over to the receptor Tim50 in the inner membrane. The membrane potential (Δψ) acts on positive charges within the presequence, which drives initial translocation through the Tim23 channel. I found that matrix targeted proteins display surprisingly different dependencies on Δψ. Interestingly, the precursor´s hypersensitivity to a reduction in Δψ was not linked to the presequence but to the mature part of the protein. The small membrane protein Pam17 is selectively recruited to the translocase by the presequence receptor Tim50 to promote transport of hypersensitive proteins. Pam17 dissociates from the import channel once the Hsp70 based import motor takes over driving the precursor in an ATP-dependent manner. I have therefore identified a second Δψ-dependent step, which acts on the mature part of the import substrate and takes place after Δψ-driven translocation of the presequence but prior to ATP-mediated import motor action. In the second part of the thesis, the molecular function of the channel forming protein Tim23 was investigated. Conserved residues in the second transmembrane segment that face the water filled pore were analyzed. Different mutations of these residues led to reduced cation selectivity and response to presequences of the Tim23 protein and renders the channel insensitive to substrates. One of these mutations, a N150A substitution, caused a growth and import defect in yeast. Since stability and assembly of the mutant Tim23 protein were not compromised, it was concluded that highly conserved residues in the channel are crucial for substrate affinity in vitro and for protein import in vivo.

Keywords: Mitochondria, Protein import, Presequence, TIM23