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Targeting of a tail-anchored protein to endoplasmic reticulum and mitochondrial outer membrane by independent but

N Borgese1, I Gazzoni, M Barberi

  • 1Consiglio Nazionale delle Ricerche, Cellular and Molecular Pharmacology Center and Department of Medical Pharmacology, University of Milan, Milan, Italy. Nica@csfic.mi.cnr.it

Molecular Biology of the Cell
|August 22, 2001
PubMed

Insights

Tail-anchored proteins target the mitochondrial outer membrane (MOM) via C-terminal arginines. This targeting occurs directly from the cytosol, not through the endoplasmic reticulum (ER).

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Many mitochondrial outer membrane (MOM) proteins are tail-anchored (TA) proteins.
  • TA proteins possess a transmembrane domain near their C terminus and an N-terminal cytosolic moiety.
  • The precise targeting mechanisms for TA proteins, particularly discrimination between MOM and endoplasmic reticulum (ER), remain unclear, though C-terminal charged residues are implicated.

Purpose of the Study:

  • To investigate the molecular determinants governing the targeting of tail-anchored proteins to the mitochondrial outer membrane (MOM).
  • To elucidate whether TA protein targeting to MOM occurs post-translationaly from the cytosol or via translocation through the endoplasmic reticulum (ER).
  • To understand the basis for dual localization of some TA proteins between MOM and ER.

Main Methods:

  • Utilized mammalian cytochrome b(5), a TA protein with known MOM and ER localized isoforms.
  • Introduced specific C-terminal residue substitutions (arginine, threonine) in cytochrome b(5) and green fluorescent protein reporter constructs.
  • Employed N-glycosylation tagging to differentiate between direct cytosolic targeting and ER-mediated translocation for MOM-targeted constructs.

Main Results:

  • Substitution of C-terminal residues with one or two arginines directed proteins to the MOM.
  • A single C-terminal threonine resulted in promiscuous localization to both MOM and ER.
  • In vivo analysis of N-glycosylation-tagged constructs showed exclusive MOM localization and minimal glycosylation, indicating direct cytosolic targeting.
  • The small fraction of glycosylated protein found in the ER was not a precursor to the unglycosylated MOM form.

Conclusions:

  • C-terminal charged residues, specifically arginines, are critical for directing tail-anchored proteins to the mitochondrial outer membrane.
  • Targeting of TA proteins to the MOM occurs directly from the cytosol, independent of the endoplasmic reticulum.
  • The endoplasmic reticulum and mitochondrial outer membrane compete for the same polypeptide pool, explaining the dual localization observed for certain TA proteins.

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