Small viral channels as tools to understand sorting of membrane proteins
Final Report Abstract
The small, similar viral encoded K+ channels Kcv and Kesv are sorted to different cellular locations. Kcv is trafficked in mammalian cells and in yeast via the secretory pathway to the plasma membrane whereas Kesv is targeted to the mitochondria. Here we examine the role of the two transmembrane domains in sorting. We previously reported that insertion of >2 hydrophobic amino acids near the end of the c-terminal transmembrane domain (TMD2) targets Kesv to the plasma membrane. We show that mutations, which make the Kesv TMD2 more similar to the Kcv channels, have no effect on sorting. After randomizing the amino acids in the TMD2 terminal end, i.e., the region, which responds to the insertion of the hydrophobic amino acids, we used a yeast complementation assay to identify two mutants that now target Kesv to the plasma membrane. The critical mutations lower the TMD2 local hydrophobicity and do not elongate the membrane-spanning domain. This is very different from Kesv mutants that sort to the plasma membrane after inserting hydrophobic amino acids into TMD2, which presumably increase the TMD2 length. Collectively, the data confirm the contribution of the Kesv C-terminal TMD2 in sorting. However, neither the length nor the hydrophobicity of TMD2 is completely responsible for the differential sorting of the small Kesv channel protein. A combined bioinformatics and mutational analyses point to the importance of a distinct structural domain at the end of TMD2 that determines Kesv sorting. By creating Kcv/Kesv chimaeras it occurred that also the n-terminal transmembrane domain (TMD1) contains a distinct sorting signal. The cellular localization of chimera with different relative portions of the two channels suggests that the default pathway for targeting of small viral channels is the secretory pathway. The Kesv channel requires the major part of the protein for a successful sorting to the mitochondria. The data identify a critical narrow domain of two amino acids in TMD1 of Kesv, which decides on a switch in mitochondrial versus plasma membrane targeting. Most interesting is a chimera, which shows by itself no sorting in cells. Only when the chimera is expressed together with the fluorescent reporter protein mKate2 it is co-sorted with the latter; depending on the targeting of the mKate2 protein the channel chimera is then sorted to the secretory pathway or to the mitochondria. Such a co-sorting of a protein without an endogenous sorting signal into the mitochondria discloses a new cellular mechanism of mitochondrial sorting.
Publications
- (2009) Selection of inhibitor-resistant viral potassium channels identifies a selectivity filter site that affects barium and amantadine block. PLosOne 4:e7496
Chatelain, F.C., Gazzarrini, S., Fujiwara, Y., Arrigoni, C., Domigan, C., Ferrara, G., Pantoja, C., Thiel, G., Moroni, A., Minor, D.L.
- (2012) The Relevance of Lysine Snorkeling in the Outer Transmembrane Domain of Small Viral Potassium Ion Channels. Biochemistry 51:5571-5579
Gebhardt, M., Henkes, L., Tayefeh, S., Hertel, B., Greiner, T., Van Etten, J., Baumeister, D., Cosentino, C., Moroni, A., Kast, S., Thiel, G.
(See online at https://doi.org/10.1021/bi3006016) - (2013) Potassium ion channels: could they have evolved from viruses? Plant Physiol. 162:1215- 1224
Thiel, G., Moroni, A., Blanc, G., Van Etten, J.L.
(See online at https://doi.org/10.1104/pp.113.219360) - Sorting of membrane proteins: Influence of N-terminal signals using the example of small viral potassium channels, Dissertation 2013, Technische Universität Darmstadt
von Chappuis, C.
- The outer transmembrane domain of the Kesv channel determines its intracellular localization. A molecular and microscopic analysis of protein sorting, Dissertation 2013, Technische Universität Darmstadt
Guthmann, T.
