Sarl is a small GTPase involved in COPⅡ vesicle transport. Previous studies showed that H79G mutation of Sarl can lock Sarl in its GTP-bound active conformation, stabilize coat assembly, and prevent the disassembly o...Sarl is a small GTPase involved in COPⅡ vesicle transport. Previous studies showed that H79G mutation of Sarl can lock Sarl in its GTP-bound active conformation, stabilize coat assembly, and prevent the disassembly of COP Ⅱ vesicle coats by reducing Sec23/24 GAP- stimulated hydrolysis. We show here that the replacement of His79 by glycine induces a large conformation change in switch Ⅱ and results in the lost of hydrogen bond between His79 and its associated nucleophilic water molecule that was hypothesized to reduce the GAP-stimulated hydrolysis during the COP Ⅱ assembly and disassembly. These results confirm that the switch Ⅱ conformation is important for COP Ⅱ disassembly through coat-controlled GTP hydrolysis.展开更多
Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix.Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too ...Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix.Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum（ER） to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site（ERES） protein Tango1.Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically：PH4 a EFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and Rab X3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast,led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco（tbc）, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.展开更多
基金This work was supported by the Frontier Interdisciplinary Innovation Fund from Fujian Institute of Research on the Structure of Matter, and Hundred Talents Project of Chinese Academy of Sciences
文摘Sarl is a small GTPase involved in COPⅡ vesicle transport. Previous studies showed that H79G mutation of Sarl can lock Sarl in its GTP-bound active conformation, stabilize coat assembly, and prevent the disassembly of COP Ⅱ vesicle coats by reducing Sec23/24 GAP- stimulated hydrolysis. We show here that the replacement of His79 by glycine induces a large conformation change in switch Ⅱ and results in the lost of hydrogen bond between His79 and its associated nucleophilic water molecule that was hypothesized to reduce the GAP-stimulated hydrolysis during the COP Ⅱ assembly and disassembly. These results confirm that the switch Ⅱ conformation is important for COP Ⅱ disassembly through coat-controlled GTP hydrolysis.
基金funded by grants from the Natural Science Foundation of China (Nos.31771600 and 31750410689)Tsinghua Initiative Program (No.20131089281)a 1000 Talents award,all to J.C.P.-P
文摘Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix.Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum（ER） to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site（ERES） protein Tango1.Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically：PH4 a EFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and Rab X3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast,led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco（tbc）, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.
