Abstract
Tail-anchored (TA) protein synthesis at the endoplasmic reticulum (ER) represents a distinct and novel process that provides a paradigm for understanding post-translational membrane insertion in eukaryotes. The major route for delivering TA proteins to the ER requires both ATP and one or more cytosolic factors that facilitate efficient membrane insertion. Until recently, the identity of these cytosolic components was elusive, but two candidates have now been suggested to promote ATP-dependent TA protein integration. The first is the cytosolic chaperone complex of Hsp40/Hsc70, and the second is a novel ATPase denoted Asna-1 or TRC40. In this study we focus on the role of the Hsp40/Hsc70 complex in promoting TA protein biogenesis at the ER. We show that the membrane integration of most TA proteins is stimulated by Hsp40/Hsc70 when using purified components and a reconstituted system. In contrast, when both Hsp40/Hsc70 and Asna-1/TRC40 are provided as a complete system, small molecule inhibition of Hsp40/Hsc70 indicates that only a subset of TA proteins are obligatory clients for this chaperone-mediated delivery route. We show that the hydrophobicity of the TA region dictates whether a precursor is delivered to the ER via the Hsp40/Hsc70 or Asna-1/TRC40-dependent route, and we conclude that these distinct cytosolic ATPases are responsible for two different ATP-dependent pathways of TA protein biogenesis.
Highlights
Tail-anchored (TA)2 proteins are a distinct class of integral membrane proteins defined by the C-terminal location of their subcellular targeting signal [1]
We show that the hydrophobicity of the TA region dictates whether a precursor is delivered to the endoplasmic reticulum (ER) via the Hsp40/Hsc70 or Asna-1/TRC40-dependent route, and we conclude that these distinct cytosolic ATPases are responsible for two different ATP-dependent pathways of TA protein biogenesis
It has been apparent for some time that TA protein biosynthesis at the ER is quite distinct from the classical signal recognition particle (SRP)-dependent route used by the majority of membrane proteins
Summary
Chemicals and Compounds—Small molecule Hsc/Hsp chaperone modulators used in this study were synthesized, as described previously [19, 20], and were solubilized in DMSO to a final concentration of 30 mM. Reconstitution of ER Integration—Ribosome-nascent chain complexes (RNCs) were generated by translating transcripts lacking a stop codon for 15 min in rabbit reticulocyte lysate (RRL) in the presence of [35S]methionine, according to the manufacturer’s instructions (Promega). In this case, the lysate was pre-spun for 3 min at 100,000 ϫ g to remove trace amounts of ER membrane. The translation reaction was treated with puromycin (1 mM) and immediately split, and the small molecule inhibitors solubilized in DMSO or DMSO alone were added at the desired concentration (5% of final volume) with subsequent incubation at 30 °C for 20 min. Post-tests were only performed when the analysis of a variance test showed a significant difference (p Ͻ 0.05) between groups. * indicates p Ͻ 0.05, and ** indicates p Ͻ 0.01 throughout
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