In Vivo Mitochondrial Import: A COMPARISON OF LEADER SEQUENCE CHARGE AND STRUCTURAL RELATIONSHIPS WITH THE IN VITRO MODEL RESULTING IN EVIDENCE FOR CO-TRANSLATIONAL IMPORT

Li Ni,Thomas S Heard,Henry Weiner

doi:10.1074/jbc.274.18.12685

Li Ni, Thomas S Heard + Show 1 more

Open Access

https://doi.org/10.1074/jbc.274.18.12685

Copy DOI

Abstract

The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments. Leader mutations that reduced the efficiency of import resulted in a cytosolic accumulation of a truncated GFP chimera that was fluorescent but devoid of a mitochondrial leader. The native leader efficiently imported before GFP could achieve a stable, import-incompetent structure, suggesting that import was coupled with translation. As a test for a co-translational mechanism, a chimera of GFP that contained the native leader of aldehyde dehydrogenase attached at the N terminus and a C-terminal endoplasmic reticulum targeting signal attached to the C terminus of GFP was constructed. This chimera was localized exclusively to mitochondria. The import result with the dual signal chimera provides support for a co-translational mitochondrial import pathway.

Highlights

The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments
Materials—The pEGFP-N1 vector and anti-GFP1 monoclonal antibody were purchased from CLONTECH; Dulbecco’s modified Eagle’s medium, calf serum, and trypsin-EDTA were obtained from Life Technologies, Inc.; the antibiotics and HEPES were purchased from Sigma; paraformaldehyde was from Aldrich; the SuperFect Transfection Reagent was obtained from QIAGEN; the TNT coupled transcription and translation system and the pGEM-7Zf(ϩ) vector were obtained from Promega; [35S] methionine and [␣-35S]dATP were purchased from Amersham Pharmacia Biotech; the SequiTherm EXCEL DNA Sequencing kit was from Epicentre Technologies Company; the alkaline phosphatase-conjugated goat anti-mouse IgG was from Bio-Rad; nitrocellulose membranes were from Schleicher and Schuell; and the Complete protease inhibitor mixture was from Roche Molecular Biochemicals
Detection of Fluorescence in HeLa Cells Transfected with EGFP and the SP1 Constructs of pALDH-EGFP—Cells that were transiently expressing EGFP displayed an observable fluorescence that was distributed throughout the whole cell, including the nucleus (Fig. 2), as shown by others [32]

Summary

In Vivo Mitochondrial Import

A COMPARISON OF LEADER SEQUENCE CHARGE AND STRUCTURAL RELATIONSHIPS WITH THE IN VITRO MODEL RESULTING IN EVIDENCE FOR CO-TRANSLATIONAL IMPORT*. The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. Mutations designed to study the role of positive charge, structure, or hydrophobicity of leader sequences have been performed under in vitro conditions (14 –19) In this setting, numerous cytosolic components that assist the import process may be absent or in limited quantity due to the conditions of the assay. There is an apparent preference of the leader for the lipid composition found in mitochondria, potential nonspecific interactions with membrane surfaces of other organelles may hinder the process of locating mitochondria, especially in mutants designed to study the import properties of the signal. Out of the diversity among all mitochondrial leader sequences, positive charges, structural motif, and the hydrophobicity of the leader may be balanced to minimize nonspecific membrane binding that may not be detected in vitro [15]

EXPERIMENTAL PROCEDURES

Charge and Structural Requirements for in Vivo Import

RESULTS

DISCUSSION