Targeting of the mitochondrial membrane proteins to the cell surface for functional studies

Hossein Ardehali, Tian Xue, Peihong Dong, Carolyn E Machamer

Research output: Contribution to journalArticle

Abstract

Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K+ and Na+ ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.

Original languageEnglish (US)
Pages (from-to)1143-1151
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume338
Issue number2
DOIs
StatePublished - Dec 16 2005

Fingerprint

Mitochondrial Proteins
Mitochondrial Membranes
Membrane Proteins
Cell membranes
Ions
Proteins
Clamping devices
Protein Sorting Signals
Cell culture
Cell Membrane
Adenosine Triphosphate
Cells
Membranes
Organelles
Membrane Potentials
Substrates
Cell Culture Techniques
Experiments

Keywords

  • ATP-binding cassette proteins
  • Membrane proteins
  • Mitochondria
  • Mitochondrial targeting signal
  • Protein trafficking

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Targeting of the mitochondrial membrane proteins to the cell surface for functional studies. / Ardehali, Hossein; Xue, Tian; Dong, Peihong; Machamer, Carolyn E.

In: Biochemical and Biophysical Research Communications, Vol. 338, No. 2, 16.12.2005, p. 1143-1151.

Research output: Contribution to journalArticle

@article{fdd41d9456e84cb586256717ee4d81a2,
title = "Targeting of the mitochondrial membrane proteins to the cell surface for functional studies",
abstract = "Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K+ and Na+ ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.",
keywords = "ATP-binding cassette proteins, Membrane proteins, Mitochondria, Mitochondrial targeting signal, Protein trafficking",
author = "Hossein Ardehali and Tian Xue and Peihong Dong and Machamer, {Carolyn E}",
year = "2005",
month = "12",
day = "16",
doi = "10.1016/j.bbrc.2005.10.070",
language = "English (US)",
volume = "338",
pages = "1143--1151",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Targeting of the mitochondrial membrane proteins to the cell surface for functional studies

AU - Ardehali, Hossein

AU - Xue, Tian

AU - Dong, Peihong

AU - Machamer, Carolyn E

PY - 2005/12/16

Y1 - 2005/12/16

N2 - Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K+ and Na+ ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.

AB - Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K+ and Na+ ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.

KW - ATP-binding cassette proteins

KW - Membrane proteins

KW - Mitochondria

KW - Mitochondrial targeting signal

KW - Protein trafficking

UR - http://www.scopus.com/inward/record.url?scp=27744457023&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=27744457023&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2005.10.070

DO - 10.1016/j.bbrc.2005.10.070

M3 - Article

C2 - 16259955

AN - SCOPUS:27744457023

VL - 338

SP - 1143

EP - 1151

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 2

ER -