Protein Import into the Yeast Endoplasmic Reticulum: Methods

Abstract

Nascent secreted proteins and integral membrane proteins in eukaryotes must be imported, or ‘translocated’ into the endoplasmic reticulum (ER). Because approximately one‐third of all cellular proteins fall into these protein classes, the translocation machinery in the ER must operate with high efficiency and accommodate a wide variety of chemically diverse substrates. Moreover, proteins can translocate into the ER either cotranslationally or posttranslationally, and each of these modes of protein entry into the ER requires unique components. Through the years, the composition and function of the ER translocation machinery was defined by the development of in vitro assays in which protein translocation can be recapitulated. For example, the posttranslational translocation of a model protein into the yeast ER can be measured using ER‐derived microsomes or reconstituted proteoliposomes. The model protein for this assay is the mating pheromone precursor, prepro‐alpha factor.

Key Concepts:

  • A large number and variety of proteins must be imported, or ‘translocated’ into the ER.

  • Proteins can enter the ER either cotranslationally, that is during synthesis, or posttranslationally, that is after synthesis.

  • The translocation reaction requires a multiprotein complex and associated factors which reside in the cytoplasm, in the ER membrane and in the ER lumenal space.

  • Posttranslational translocation can be recapitulated using ER‐derived microsomes prepared from the yeast Saccharomyces cerevisiae, or using reconstituted proteoliposomes into which the translocation machinery has been integrated.

  • The in vitro reaction has helped to define how components of the translocation machinery function.

  • The translocated substrate can be mutated to assess how aberrant proteins are targeted for ER‐associated degradation (also known as ERAD).

Keywords: translocation; endoplasmic reticulum; yeast; sec61; microsomes; ERAD

Figure 1.

The products after an in vitro posttranslational translocation assay are divided into thirds. In tube 1, the reaction is left untreated, whereas in tube 2 exogenous trypsin is added to digest untranslocated material. In tube 3, both trypsin and TritonX‐100 are added to digest both the translocated and untranslocated material. Note: A small amount of residual ppαf and pαf remain in this tube due to the presence of protein aggregates (see Figure ).

Figure 2.

Translocation of ppαf in vitro.

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Further Reading

Schnell DJ and Hebert DN (2003) Protein translocons: multifunctional mediators of protein translocation across membranes. Cell 112: 491–505.

Wickner W and Schekman R (2005) Protein translocation across biological membranes. Science 310: 1452–1456.

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Brodsky, Jeffrey L, and Morrow, Michael W(Sep 2010) Protein Import into the Yeast Endoplasmic Reticulum: Methods. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002619.pub3]