Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER.

Sec12p is an integral membrane protein required in vivo and in vitro for the formation of transport vesicles generated from the ER. Vesicle budding and protein transport from ER membranes containing normal levels of Sec12p is inhibited in vitro by addition of microsomes isolated from a Sec12p-overproducing strain. Inhibition is attributable to titration of a limiting cytosolic protein. This limitation is overcome by addition of a highly enriched fraction of soluble Sar1p, a small GTP-binding protein, shown previously to be essential for protein transport from the ER and whose gene has been shown to interact genetically with sec12. Furthermore, Sar1p binding to isolated membranes is enhanced at elevated levels of Sec12p. Sar1p-Sec12p interaction may regulate the initiation of vesicle budding from the ER.

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Reconstitution in vitro of a membrane-fusion event involved in constitutive exocytosis. A role for cytosolic proteins and a GTP-binding protein, but not for Ca2+

Biochemical Journal ◽

10.1042/bj2850383 ◽

1992 ◽

Vol 285 (2) ◽

pp. 383-385 ◽

Cited By ~ 6

Author(s):

J M Edwardson ◽

P U Daniels-Holgate

Keyword(s):

Membrane Fusion ◽

Binding Protein ◽

Fusion Event ◽

Gtp Binding Protein ◽

In Vitro System ◽

Cytosolic Proteins ◽

Golgi Transport ◽

Gtp Binding ◽

Transport Vesicles

The fusion of post-Golgi transport vesicles with the plasma membrane is perhaps the least well understood step in the network of intracellular membrane traffic. We have used an ‘in vitro’ system to study this membrane-fusion event. We show here that fusion requires the presence of cytosolic proteins, but not Ca2+, and is inhibited by the non-hydrolysable GTP analogue guanosine 5′-[gamma-thio]triphosphate, which indicates the involvement of a GTP-binding protein.

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The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants.

The Journal of Cell Biology ◽

10.1083/jcb.109.3.1015 ◽

1989 ◽

Vol 109 (3) ◽

pp. 1015-1022 ◽

Cited By ~ 132

Author(s):

R A Bacon ◽

A Salminen ◽

H Ruohola ◽

P Novick ◽

S Ferro-Novick

Keyword(s):

Golgi Apparatus ◽

Binding Protein ◽

Yeast Cells ◽

Carboxypeptidase Y ◽

Loss Of Function ◽

Gtp Binding Protein ◽

Gtp Binding ◽

Transport Defect

The YPT1 gene encodes a raslike, GTP-binding protein that is essential for growth of yeast cells. We show here that mutations in the ypt1 gene disrupt transport of carboxypeptidase Y to the vacuole in vivo and transport of pro-alpha-factor to a site of extensive glycosylation in the Golgi apparatus in vitro. Two different ypt1 mutations result in loss of function of the Golgi complex without affecting the activity of the endoplasmic reticulum or soluble components required for in vitro transport. The function of the mutant Golgi apparatus can be restored by preincubation with wild-type cytosol. The transport defect observed in vitro cannot be overcome by addition of Ca++ to the reaction mixture. We have also established genetic interactions between ypt1 and a subset of the other genes required for transport to and through the Golgi apparatus.

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Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1505995112 ◽

2015 ◽

Vol 112 (28) ◽

pp. E3679-E3688 ◽

Cited By ~ 39

Author(s):

Susanne de Boor ◽

Philipp Knyphausen ◽

Nora Kuhlmann ◽

Sarah Wroblowski ◽

Julian Brenig ◽

...

Keyword(s):

Binding Protein ◽

Rat Tissues ◽

Lysine Acetylation ◽

Nucleotide Exchange ◽

Gtp Binding Protein ◽

Cellular Effects ◽

Gtp Binding ◽

Cytoplasmic Transport

Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed.

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Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

Molecular Biology of the Cell ◽

10.1091/mbc.e08-09-0905 ◽

2009 ◽

Vol 20 (5) ◽

pp. 1388-1399 ◽

Cited By ~ 117

Author(s):

Mike Ngo ◽

Neale D. Ridgway

Keyword(s):

Secretory Pathway ◽

Protein Transport ◽

Binding Protein ◽

Dominant Negative ◽

Ph Domain ◽

Oxysterol Binding Protein ◽

Large Gene ◽

Dominant Negative Variant

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER–Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P–dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN.

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Crystal structure of the GTP-binding protein-like domain of AGAP1

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x21003150 ◽

2021 ◽

Vol 77 (4) ◽

Author(s):

Nuo Cheng ◽

Hao Zhang ◽

Shiyan Zhang ◽

Xiaodan Ma ◽

Guoyu Meng

Keyword(s):

Membrane Trafficking ◽

Protein Transport ◽

Structural Information ◽

Binding Protein ◽

Lymphoblastic Leukemia ◽

Diffusion Method ◽

Gtp Binding Protein ◽

Aberrant Expression ◽

Cell Parameters ◽

Gtp Binding

AGAP1 is often considered to regulate membrane trafficking, protein transport and actin cytoskeleton dynamics. Recent studies have shown that aberrant expression of AGAP1 is associated with many diseases, including neurodevelopmental disorders and acute lymphoblastic leukemia. It has been proposed that the GTP-binding protein-like domain (GLD) is involved in the binding of cofactors and thus regulates the catalytic activity of AGAP1. To obtain a better understanding of the pathogenic mechanism underpinning AGAP1-related diseases, it is essential to obtain structural information. Here, the GLD (residues 70–235) of AGAP1 was overexpressed in Escherichia coli BL21 (DE3) cells. Affinity and gel-filtration chromatography were used to obtain AGAP1GLD with high purity for crystallization. Using the hanging-drop vapor-diffusion method with the protein at a final concentration of 20 mg ml−1, AGAP1GLD protein crystals of suitable size were obtained. The crystals were found to diffract to 3.0 Å resolution and belonged to space group I4, with unit-cell parameters a = 100.39, b = 100.39, c = 48.08 Å. The structure of AGAP1GLD exhibits the highly conserved functional G1–G5 loops and is generally similar to other characterized ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), implying an analogous function to Arf GAPs. Additionally, this study indicates that AGAP1 could be classified as a type of NTPase, the activity of which might be regulated by protein partners or by its other domains. Taken together, these results provide insight into the regulatory mechanisms of AGAP1 in cell signaling.

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The synthesis of murine ferrochelatase in vitro and in vivo

Biochemical Journal ◽

10.1042/bj2540799 ◽

1988 ◽

Vol 254 (3) ◽

pp. 799-803 ◽

Cited By ~ 29

Author(s):

S R Karr ◽

H A Dailey

Keyword(s):

Membrane Protein ◽

Biosynthetic Pathway ◽

Integral Membrane Protein ◽

Mitochondrial Proteins ◽

Isolated Mitochondria ◽

Mitochondrial Inner Membrane ◽

Carbonyl Cyanide ◽

A Cell

Ferrochelatase (protohaem ferro-lyase, EC 4.99.1.1), the terminal enzyme of the haem-biosynthetic pathway, is an integral membrane protein of the mitochondrial inner membrane. When murine erythroleukaemia cells are labelled in vivo with [35S]methionine, lysed, and the extract is immunoprecipitated with rabbit anti-(mouse ferrochelatase) antibody, a protein of Mr 40,000 is isolated. However, when isolated mouse RNA is translated in a cell-free reticulocyte extract, a protein of Mr 43,000 is isolated. Incubation of this Mr 43,000 protein with isolated mitochondria resulted in processing of the Mr 43,000 precursor to the Mr 40,000 mature-sized protein. Addition of carbonyl cyanide m-chlorophenylhydrazone and/or phenanthroline inhibits this processing. These data indicate that ferrochelatase, like most mitochondrial proteins, is synthesized in the cytoplasm as a larger precursor and is then translocated and processed to a mature-sized protein in an energy-required step.

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Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport.

The Journal of Cell Biology ◽

10.1083/jcb.114.4.671 ◽

1991 ◽

Vol 114 (4) ◽

pp. 671-679 ◽

Cited By ~ 47

Author(s):

T Oka ◽

S Nishikawa ◽

A Nakano

Keyword(s):

Temperature Sensitivity ◽

Protein Function ◽

Secretory Pathway ◽

Protein Transport ◽

Temperature Sensitive ◽

Transport Reaction ◽

Golgi Transport ◽

Gtp Binding

In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.

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Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.135.3.585 ◽

1996 ◽

Vol 135 (3) ◽

pp. 585-595 ◽

Cited By ~ 90

Author(s):

M J Kuehn ◽

R Schekman ◽

P O Ljungdahl

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Membrane Protein ◽

Integral Membrane Protein ◽

Plasma Membrane Atpase ◽

Amino Acid Permease ◽

Transport Vesicles ◽

Amino Acid Permeases ◽

Histidine Permease

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

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