3. (6pts ea GDP. Plea provide a brief explanation (2 sentences max). cnj Suppose
ID: 261588 • Letter: 3
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3. (6pts ea GDP. Plea provide a brief explanation (2 sentences max). cnj Suppose the following GTPases are mutated so that they permanently bind to se predict the location where the stated cargo proteins would accumulate, and A. GTPase: Ran, Cargo: Protein with NLS sequence Cargo protein location with mutant Ran- Explanation - B. GTPase: Sar1, Cargo: Secreted protein Cargo protein location with mutant Sar1- Explanation - C. GTPase: Dynamin, Cargo: Low Density Lipoprotein (LDL) particle Cargo protein location with mutant dynamin - Explanation - D. GTPase: ARF1, Cargo: ER Resident protein with KDEL sequence Cargo protein location with mutant ARF1- Explanation -Explanation / Answer
A - Cargo protein located with mutant ran with NLS sequece :
A nuclear localization sequence (NLS) is an amino acid sequence that tags a protein for import into the cell nucleus by nuclear transport.
The transport of some small protein cargoes through the nuclear pore complex (NPC) can occur in vitro in the absence of nucleoside triphosphate hydrolysis.
We now demonstrate that in the importin ?/? and transportin import pathways, efficient in vitro transport of large proteins, in contrast to smaller proteins, requires hydrolyzable GTP and the small GTPase Ran.
Morphological and biochemical analysis indicates that the presence of Ran and GTP allows large cargo to efficiently cross central regions of the NPC.
We further demonstrate that this function of RanGTP at least partly involves its direct binding to importin ? and transportin.
We suggest that RanGTP functions in these pathways to promote the transport of large cargo by enhancing the ability of import complexes to traverse diffusionally restricted areas of the NPC.
B - Cargo proteins located with mutant sar-1 :
Small GTPase component of the coat protein complex II (COPII) which promotes the formation of transport vesicles from the endoplasmic reticulum (ER).
The coat has two main functions, the physical deformation of the endoplasmic reticulum membrane into vesicles and the selection of cargo molecules. SAR1 controls the coat assembly in a stepwise manner.
Activated SAR1-GTP by SEC12 binds to membranes first and recruits the SEC23/24 complex.
These SEC23/24-SAR1 prebudding intermediates are then collected by the SEC13/31 complex as subunits polymerize to form coated transport vesicles.
Conversion to SAR1-GDP triggers coat release and recycles COPII subunits.
C - Cargo proteins located with mutant dynamic: LDL
In mammals, water-insoluble cholesterol circulates in the form of low density lipoprotein (LDL) particles, and must be internalized and de-esterified to enable entry into cellular membrane pools.
Mobilization of LDL-derived cholesterol involves receptor-mediated endocytosis of the LDL particle, followed by swift recycling of the transmembrane receptor from endosomes back to the cell surface, but with synchronous movement of the disengaged LDL carrier particle to the lysosome for catabolic processing.
Establishing the molecular details of tightly regulated LDL receptor movement within cells has illuminated many fundamental aspects of intracellular vesicular trafficking and, significantly, is tightly associated with human health and disease.
D - Cargo proteins located with mutant ARF1 : ER resident protein with KDEL sequence :
KDEL receptor ERD2 is sorted into COPI vesicles for Golgi-to-ER transport is largely unknown.
Interactions between proteins of the COPI transport machinery occurring during a wave of transport of a KDEL ligand were studied in living cells.
FRET between CFP and YFP fusion proteins was measured by multifocal multiphoton microscopy and bulk-cell spectrofluorimetry.
Ligand binding induces oligomerization of ERD2 and recruitment of ARFGAP to the Golgi, where the (ERD2)n/ARFGAP complex interacts with membrane-bound ARF1.
During KDEL ligand transport, interactions of ERD2 with beta-COP and p23 decrease and the proteins segregate.
Both p24a and p23 interact with ARF1, but only p24 interacts with ARFGAP.
These findings suggest a model for how cargo-induced oligomerization of ERD2 regulates its sorting into COPI-coated buds.
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