Browsing by Subject "Membrane Protein"
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Item Investigating cotranslational protein integration into the endoplasmic reticulum membrane(Texas A&M University, 2005-02-17) McCormick, Peter JosephDuring co-translational integration, the transmembrane (TM) sequence of a nascent membrane protein moves laterally into the ER lipid bilayer upon reaching the translocon. Our lab has previously shown that this movement is a multistep process, but it was not clear whether the observed photocrosslinking of the TM segment to translocon proteins resulted from specific interactions or simply from TM-translocon proximity. If the latter, the TM α-helix will be oriented randomly with respect to translocon proteins, whereas, if the former, a specific TM helix surface would face TRAM and/or Sec61α. Integration intermediates were prepared by in vitro translation of truncated mRNAs in the presence of a Lys-tRNA analog with a photoreactive moiety attached to the lysine side-chain. When photoadduct formation was monitored as a function of probe location within the TM α-helix, we found that the extent of photocrosslinking to TRAM and Sec61α was non-random. Thus, the TM sequence occupies a distinct location within the translocon, a result that can only be achieved through protein-protein interactions that mediate the lateral movement, positioning, and integration of the TM sequence. In the case of multi-spanning membrane proteins, it was unknown how multiple hydrophobic regions integrated into the ER membrane. By placing photoprobes within each of several TM domains of a multi-spanning membrane protein, we were able to determine at what stage of integration each TM segment was no longer adjacent to translocon proteins. Using this approach we were able to establish a mechanism of integration for multi-spanning membrane proteins co-translationally inserted into the ER membrane.Item Trafficking of integral membrane proteins of the inner nuclear membrane can be mediated by the ''sorting motif'' of autographa californica nucleopolyhedrovirus odv-e66(Texas A&M University, 2006-10-30) Williamson, Shawn TThe amino-terminal 33 amino acids of the baculovirus integral membrane protein, ODV-E66, are sufficient for localization of fusion proteins to viralinduced intranuclear microvesicles (MV) and occlusion derived virus envelopes during infection, and has been termed the sorting motif (SM). When abundantly expressed, SM-fusions are also detected in the inner nuclear membrane (INM), outer nuclear membrane and endoplasmic reticulum of infected cells, suggesting proteins with the SM use the same trafficking pathway as cellular INM proteins to traffic to nuclear membranes. This study identifies the essential characteristics required for sorting of the SM to the INM of uninfected cells, and the MV and ODV envelopes of infected cells. These features are an 18 amino acid transmembrane sequence that lacks polar and charged amino acids (a.a.) with a cluster of charged a.a. spaced 5-11 residues from the end of the transmembrane sequence. A comparison of the a.a. sequence of these SM features with cellular INM proteins shows the features are conserved. The model of INM protein sorting and localization predicts the only known sorting event during INM protein trafficking is immobilization/retention in the INM. This study uses confocal microscopy and fluorescence recovery after photobleaching to compare the localization and mobility of lamin B receptor (LBR) fusions (which contain SM-like sequences) to a viral SM fusion when expressed in either mammalian or insect cells. The results show that immobilization is not necessarily required for accumulation of proteins in the INM. Furthermore, the results from infected cells show that an active sorting event, likely independent of immobilization, can distinguish the viral SM from cellular sequences similar to the SM. The results of this study show that sorting of proteins to the INM can be mediated by the viral SM or INM protein SM-like sequences that can function either independent of, or in addition to, immobilization. These data combined with recent reports suggest that in addition to diffusion:retention a signal mediated mechanism for sorting and localization to the INM can occur.