Browsing by Subject "Intracellular Membranes"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Ceramide-Induced Alternative Translocation of TM4SF20(2015-10-29) Lee, Ching En; Pfeiffer, Julie K.; McKnight, Steven L.; Kahn, Jeffrey; Nijhawan, Deepak; Ye, JinThe polytopic membrane protein TM4SF20 (transmembrane 4 L6 family 20) is a protein containing four transmembrane helices that inhibits the Regulated Intramembrane Proteolysis (RIP) of the transcriptional factor CREB3L1 (cAMP response element binding protein 3-like 1), a transcription factor synthesized as a membrane-bound precursor. CREB3L1 RIP is induced by several stimuli: ER stress, viral infections, the chemotherapeutic drug, doxorubicin, and the sphingolipid, ceramide. Additionally, TGF-β (transforming growth factor-β), a cytokine known to stimulate collagen production, induces the proteolytic activation of CREB3L1 in human A549 cells through inhibition of TM4SF20 expression, which normally inhibits RIP of CREB3L1. We also find that the TM4SF20 regulation of CREB3L1 RIP is regulated by ceramide. In this study we find that ceramide can regulate the ability of first transmembrane domain of TM4SF20 to determine its orientation in the membrane. Under normal conditions, TM4SF20 is synthesized as a protein that inhibits the cleavage of CREB3L1 when TRAM2 (translocation associated membrane protein 2) is associated with the ER translocon. Excess ceramide dissociates TRAM2 from the ER translocon such that the N-terminus of TM4SF20 can no longer be forced by the first transmembrane domain to function as a signal peptide. Under excess ceramide conditions, TM4SF20 adopts a completely opposite topology and allows the cleavage of CREB3L1 to proceed. We have designated this novel mechanism for transmembrane protein regulation as "alternative translocation."Item Using Advanced Fluorescence Microscopy to Analyze Intracellular Trafficking of FcRn In Live Cells(2013-01-17) Gan, Zhuo; Ward, E. Sally; Ober, Raimund J.The MHC class I-related Fc receptor (FcRn) regulates the in vivo half-life of immunoglobulin G (IgG) and transports IgG across cell barriers. The intracellular trafficking of FcRn is central to its diverse functions. FcRn, like all receptors, is transferred to lysosomes for constitutive degradation to maintain a balance between synthesis and breakdown. Using live cell imaging, a novel lysosomal delivery pathway for FcRn has been observed. Unlike signaling receptors that enter the intraluminal vesicles in late endosomes, FcRn remains on the limiting membrane of late endosomes and is delivered to lysosomes through a selective, primarily tubule-mediated process. Following transfer, FcRn is rapidly internalized into the lysosomal lumen. By contrast, LAMP1 remains on the limiting membrane of lysosomes. Rab5 can persist on late endosomes, which can not only fuse with lysosomes, but can also give rise to tubulovesicular carriers that enter the recycling pathway. Thus, late endosomes are functionally plastic. These observations have relevance to understanding lysosomal delivery pathways. A combination of MUltifocal plane Microscopy (MUM) and localized photoactivation ('LP-MUM') has been developed to investigate the intracellular recycling pathway of FcRn. LP-MUM has been used to activate photoactivatable GFP (PAGFP) tagged proteins in individual sorting endosomes within cells, followed by imaging in two focal planes simultaneously. This approach has enabled the tracking of small, motile and dense transport carriers (TCs) that deliver FcRn to different destinations within the cell. The Rab GTPases, SNX4 and APPL1 play important roles in various steps of receptor trafficking pathways, and their associations with TCs has also been investigated. Four distinct itineraries taken by TCs at various stages of FcRn recycling have been characterized. In addition, the effectors associated with TCs on different pathways have been identified. APPL1+ TCs can transfer FcRn from the plasma membrane to pre-existing sorting endosomes. Interendosomal TCs migrate between sorting endosomes and are Rab4+/SNX4+ but Rab11-/APPL1-. Post-endosomal TCs that deliver FcRn to the plasma membrane are Rab11+ but Rab4-/SNX4-/APPL1-. Unexpectedly, a novel class of 'looping' TCs that leave a sorting endosome and return to the same endosome after several minutes has also been observed. The 'looping' TCs are Rab11+/Rab4+/SNX4+. The analyses of these TCs should have general relevance to other receptors and cargo on the recycling pathway.