Download PL1 Secretion of large particles and miRNA

Plenary Lecture
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Plenary Lecture
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Secretion of large particles and miRNA
Randy Schekman |8QLYRI&DOLIRUQLD%HUNHOH\|
COPII is the universal coat for capture of cargo proteins en route to the Golgi apparatus. Although normal COPII
vesicles are fairly uniform in size, ca. 80–90 nm in diameter, certain cargo molecules, eg. procollagen and large lipoprotein
particles, are too large to be accommodated and yet the COPII coat participates in their packaging at the ER. In
collaboration with the laboratory of M. Rape, we found that ubiquitylation of the cage forming subunit of COPII, Sec31,
promotes the formation of large COPII structures that speed the transfer of procollagen out of the ER. Using advanced
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tetraspanin protein CD63, and selected microRNAs that may be conveyed between tissues to control gene expression in
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for the isolation of a unique exosome species culminating in the immunoisolation of vesicles on anti-CD63 antibody beads.
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selective incorporation of an exosomal microRNA. HEK293 cells were lysed and washed membranes sedimented from a
post-nuclear supernatant fraction were mixed with miRNA (miR-223), cytosol and ATP and incubated at 30°C. miR-223,
but not a cellular miRNA (miR-190), was incorporated into an RNase-protected (but not detergent and RNase-protected)
membrane, dependent on cytosolic proteins, ATP and incubation at physiologic temperature. To identify proteins that are
directly involved in packaging miR-223, we combined in vitro packaging with proteomics. Y-box protein I (YBX1) coprecipitated with tagged miR-223 isolated from a complete cell-free reaction. We found that cytosol from ¨YBX1 cells did
not support miR-223 packaging in vitro but that packaging was restored with cytosol from ¨YBX1 cells transfected with
<%;2XUUHVXOWVVKRZWKDWHI¿FLHQWSDFNDJLQJRIPL5in vitro and in vivo requires Y-box proteins in HEK293T cells.
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Cytoplasm and Cell Regulation
Yoshihiro Yoneda | Natl. Inst. of Biomed. Innov. (NIBio) |
In eukaryotic cells, the nucleus is separated from the cytoplasm by a double lipid bilayer called nuclear envelope. The
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consist of multiple copies of approximately 30 different proteins called nucleoporins. Protein transport through the NPCs
involves a transport factor that recognizes a nuclear localization signal (NLS) for nuclear import or a nuclear export signal
(NES) for nuclear export. Typically, a basic-type classical NLS-containing cargo is recognized by importin Į. Importin ȕ
binds to importin Į bound to the cargo to form a ternary complex. This trimer translocates through the NPC. After the
translocation, within the nucleus, GTP bound form of Ran (RanGTP) binds to importin ȕ to trigger the dissociation of the
trimer. Then, karyophilic proteins can function in the nucleus.
Nuclear transport plays crucial roles in many biological processes such as cell fate determination and intracellular signal
transduction. Importin ĮLVLGHQWL¿HGDVDF1/6UHFHSWRUPROHFXOHDQGVHYHQLPSRUWLQĮVKDYHEHHQLGHQWL¿HGLQKXPDQ
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recently demonstrated that the expression of the importin Į subtype is switched from Į2 to Į1 during neural differentiation
in mouse ES cells and that this switching has a major impact on cell differentiation. We also have shown a novel cell-fate
determination mechanism in which importin Į2 negatively regulates the nuclear import of certain transcription factors to
maintain ES cell properties. Further, we have found that Retinoblastoma Binding Protein 4, RBBP4, functions as a novel
UHJXODWRU\IDFWRUWRLQFUHDVHWKHHI¿FLHQF\RILPSRUWLQĮ/ȕ-mediated nuclear import by accelerating the release of importin
ȕ from importin Į through its competitive binding to the importin ȕ-binding (IBB) domain of importin Į in the presence of
nuclear RanGTP. We also have found that the down-regulation of RBBP4 induces cellular senescence. Thus, from these
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Nucleocytoplasmic Transport, Importin, Nuclear Pore Complex
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