p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum

PLoS Biol. 2012;10(5):e1001336. doi: 10.1371/journal.pbio.1001336. Epub 2012 May 29.

Abstract

In metazoans, the majority of mRNAs coding for secreted and membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). Although the targeting of these transcripts to the surface of the ER can be mediated by the translation of a signal sequence and their maintenance is mediated by interactions between the ribosome and the translocon, it is becoming increasingly clear that additional ER-localization pathways exist. Here we demonstrate that many of these mRNAs can be targeted to, and remain associated with, the ER independently of ribosomes and translation. Using a mass spectrometry analysis of proteins that associate with ER-bound polysomes, we identified putative mRNA receptors that may mediate this alternative mechanism, including p180, an abundant, positively charged membrane-bound protein. We demonstrate that p180 over-expression can enhance the association of generic mRNAs with the ER. We then show that p180 contains a lysine-rich region that can directly interact with RNA in vitro. Finally, we demonstrate that p180 is required for the efficient ER-anchoring of bulk poly(A) and of certain transcripts, such as placental alkaline phosphatase and calreticulin, to the ER. In summary, we provide, to our knowledge, the first mechanistic details for an alternative pathway to target and maintain mRNA at the ER. It is likely that this alternative pathway not only enhances the fidelity of protein sorting, but also localizes mRNAs to various subdomains of the ER and thus contributes to cellular organization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Alkaline Phosphatase / genetics
  • Alkaline Phosphatase / metabolism
  • Animals
  • Base Sequence
  • COS Cells
  • Calreticulin / genetics
  • Calreticulin / metabolism
  • Cell Size
  • Chlorocebus aethiops
  • Cloning, Molecular
  • Digitonin / pharmacology
  • Endoplasmic Reticulum / genetics
  • Endoplasmic Reticulum / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • GPI-Linked Proteins / genetics
  • GPI-Linked Proteins / metabolism
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • In Situ Hybridization, Fluorescence
  • Insulin / genetics
  • Insulin / metabolism
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Lentivirus / genetics
  • Lentivirus / metabolism
  • Lysine / metabolism
  • Molecular Sequence Data
  • Plasmids / genetics
  • Plasmids / metabolism
  • Protein Interaction Mapping
  • Protein Transport
  • Proteins / genetics
  • Proteins / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Ribosomes / metabolism*

Substances

  • Calreticulin
  • GPI-Linked Proteins
  • Insulin
  • Isoenzymes
  • Leydig insulin-like protein
  • Proteins
  • RNA, Messenger
  • Receptors, Cytoplasmic and Nuclear
  • ribosome receptor p180, human
  • Green Fluorescent Proteins
  • Alkaline Phosphatase
  • alkaline phosphatase, placental
  • Lysine
  • Digitonin