Guest Post: The cellular postal system: more postmen don’t spoil the delivery

Bodo Stern writes:

Remember the children’s game “Telephone” in which the first participant whispers a phrase to their neighbor who in turn whispers what they believe to have heard to the next player, and so on? The phrase announced by the final player often differs substantially and in hilarious ways from the original message. Luckily, molecular cascades are more accurate than these human chains: not only are they able to carry information faithfully, they can introduce specificity and integrate several inputs. An interesting recent discovery of such a molecular chain comes from Vlad Denic’s laboratory at the Department for Molecular and Cellular Biology at Harvard University. The work, published in Molecular Cell, identifies new components in a pathway that delivers so-called tail-anchored (TA) proteins to their destination in the endoplasmatic reticulum (ER) membrane.

The classic example of membrane protein delivery to the ER involves, of course, the signal recognition particle (SRP), which recognizes a signal sequence at the N-terminus of its cargo proteins while the protein emerges from the ribosome exit channel. This recognition event blocks further translation until the SRP-cargo-ribosome complex delivers its cargo protein to the translocation pore on the ER. But a subset of ER resident membrane proteins, the tail-anchored proteins, cannot be delivered this way: they carry the ER-targeting information and trans-membrane domain at the very C-terminus, making co-translational handover impossible. TA-proteins face another fascinating problem: they fall into two classes, one that is targeted to the ER and another that is targeted to mitochondria. How does the cell ensure correct delivery? To understand how TA-proteins can reach two distinct “postal addresses” within the cell, we first need to know who the molecular postmen are. Several proteins are known in budding yeast to be engaged in TA-protein targeting to the ER, as part of the so-called GET pathway (for “guided entry of TA-proteins”): Get1 and Get2, the heterodimeric TA-protein receptor in the ER membrane, and Get3, a cytoplasmic ATPase which delivers ER-bound TA-proteins to the Get1/2 receptor (Stefanovic and Hedge, 2007; Schuldiner et al, 2008). Two other suspects, Get4 and Get5, were found guilty by association based on genetic interaction profiles that are remarkably similar to Get1-3 (Jonikas et al., 2009).

Wang et al. now report the purification from budding yeast of a transmembrane recognition complex (TRC) that acts in between the ribosome and Get3. This complex, which contains Get4, Get5, Sgt2, and several heat-shock proteins, can distinguish between ER- and mitochondria-bound TA-proteins and selectively hands off the ER-bound TA-proteins to Get3. Sgt2 turns out to be the central TRC component, binding Get5 with its N-terminus (and indirectly to Get4 through Get 5), the heat shock proteins in the middle, and the ER-targeting domain of the TA-protein through a C-terminal methionine-rich domain.

The strength and beauty of this paper lies in the elegant use of get mutant extracts and recombinant proteins to dissect protein complex topology and function. For reasons that are not entirely clear, get deletion mutants are viable even though the GET pathway delivers critical proteins such as vesicle fusion mediators called SNARE proteins. Maybe a sufficient amount can insert spontaneously into the destination membrane without the aid of the GET pathway. In any case, mutant viability is less a concern than a powerful tool. Here is a great example: to address whether Sgt2 hands over the cargo protein to Get3, the authors in vitro translate ³⁵S-labelled TA-protein (Sec22) in an extract from yeast in which both Get3 and Get5 are deleted. They had established that Sgt2 can bind Sec22 in the absence of Get3 or Get5, making it possible to purify the Sgt2-Sec22 complex without any traces of bound Get3, 4, 5 (Get4 only binds to Sgt2 through Get5). The authors can then test, by adding combinations of recombinant Get3, 4 and 5, which GET proteins are necessary and sufficient to release Sec22 from Sgt2. It turns out that both Get4 and Get5 are necessary to transfer the TA-protein from Sgt2 to Get3. The combination of purified Sgt2-Sec22 and recombinant Get3, 4 and 5 is also sufficient to drive TA-protein membrane insertion into isolated ER membranes in an in vitro reconstitution experiment. These experiments would not be possible using, for example, wildtype extracts since the intermediate Sgt2-Sec22 complex could never be trapped and purified.

What about discrimination of ER-and mitochondria bound TA-proteins? Sgt2 selectively recognizes the ER-targeting domain of TA-proteins. A mitochondrial TA-protein, Fis1, is not efficiently inserted into ER membranes in vitro and also cannot bind directly to Sgt2 (although, curiously, it does bind to the heat shock factors of the TRC complex). Sgt2 can, however, recognize a mutant Fis1 that is known to mislocalize to the ER; this mutant Fis1 is also efficiently inserted into ER membranes in vitro. In summary, Sgt2 can discriminate ER-and mitochondria directed TA-proteins and hands over ER-bound cargo to Get3, which in turn delivers it to the ER-receptor Get1-2. It is possible that a second discrimination of the ER-signal occurs after Sgt2, at the level of Get3. If true, such double scrutiny of the specificity signal could be another example for kinetic proofreading – the same free energy difference between ER- and mitochondria-bound TA-proteins is sampled twice in a row to increase accuracy of delivery. Maybe the cellular postal system needs the vigilance of several molecular postmen to make sure that small differences in the addresses are noticed, and cargo gets delivered to the right place most of the time.

Wang F, Brown EC, Mak G, Zhuang J, & Denic V (2010). A Chaperone Cascade Sorts Proteins for Posttranslational Membrane Insertion into the Endoplasmic Reticulum. Molecular Cell PMID: 20850366