scholarly journals A Single C-terminal Peptide Segment Mediates Both Membrane Association and Localization of Lysyl Hydroxylase in the Endoplasmic Reticulum

2000 ◽  
Vol 275 (23) ◽  
pp. 17863-17868 ◽  
Author(s):  
Marko Suokas ◽  
Raili Myllylä ◽  
Sakari Kellokumpu
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Association ◽  
Lysyl Hydroxylase

Cytosolic phospholipase A2-α associates with plasma membrane, endoplasmic reticulum and nuclear membrane in glomerular epithelial cells

2000 ◽  
Vol 353 (1) ◽  
pp. 79-90 ◽  
Author(s):  
Jianhong LIU ◽  
Tomoko TAKANO ◽  
Joan PAPILLON ◽  
Abdelkrim KHADIR ◽  
Andrey V. CYBULSKY
Keyword(s):  
Endoplasmic Reticulum ◽  
Arachidonic Acid ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Phospholipase A2 ◽  
Cytosolic Phospholipase A2 ◽  
Membrane Association ◽  
Cytosolic Phospholipase ◽  
Glomerular Epithelial Cells ◽  
In Cells

Eicosanoids mediate complement-dependent glomerular epithelial injury in experimental membranous nephropathy. The release of arachidonic acid from phospholipids by cytosolic phospholipase A2 (cPLA2) is the rate-limiting step in eicosanoid synthesis. The present study examines the association of cPLA2 with membranes of organelles. Glomerular epithelial cells were disrupted by homogenization in Ca2+-free buffer; organelles were separated by gradient centrifugation. The distribution of cPLA2 and organelles was analysed by immunoblotting with antibodies against cPLA2 and organelle markers, or by enzyme assay. In cells incubated with or without the Ca2+ ionophore ionomycin plus PMA, cPLA2 co-localized with plasma membrane, endoplasmic reticulum and nuclei, but not with mitochondria or Golgi. A greater amount of cPLA2 was associated with membranes in stimulated cells, but membrane-associated cPLA2 was readily detectable under resting conditions. The pattern of association of cPLA2 with membrane in cells treated with antibody and complement was similar to that in cells stimulated with ionomycin plus PMA; however, complement did not enhance the membrane association of cPLA2 protein. To determine the functional role of membrane association of cPLA2, phospholipids were labelled with [3H]arachidonic acid. Cells were then incubated with or without antibody and complement and were fractionated. Complement induced a loss of radioactivity from the plasma membrane, endoplasmic reticulum and nuclei, but not from the mitochondrial fraction. Thus the release of arachidonic acid by cPLA2 is due to the hydrolysis of phospholipids at multiple subcellular membrane sites, including the endoplasmic reticulum, plasma membrane and nucleus.

scholarly journals Membrane interactions of simian virus 40 large T-antigen: influence of protein sequences and fatty acid acylation.

1984 ◽  
Vol 4 (8) ◽  
pp. 1542-1550 ◽  
Author(s):  
U Klockmann ◽  
M Staufenbiel ◽  
W Deppert
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Protein Sequences ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Membrane Association ◽  
Large T Antigen ◽  
Amino Terminal

To sort out possible influences of protein sequences and fatty acid acylation on the plasma membrane association of simian virus 40 large T-antigen, we have analyzed the membrane interactions of carboxy-terminal fragments of large T-antigen, encoded by the adenovirus type 2 (Ad2+)-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2. The 28,000 (28K)-molecular-weight protein of Ad2+ND1 as well as the 42K and 56K proteins of Ad2+ND2 associate preferentially with membranous structures and were found in association with the membrane system of the endoplasmic reticulum and with plasma membranes. Neither the endoplasmic reticulum membrane- nor the plasma membrane-associated 28K protein of Ad2+ND1 is fatty acid acylated. We, therefore, conclude that fatty acid acylation is not necessary for membrane association of this protein and suggest that an amino acid sequence in this protein is responsible for its membrane interaction. In contrast, the 42K and 56K proteins of Ad2+ND2 in plasma membrane fractions contain fatty acid. However, the interaction of these proteins with the plasma membrane differs from that of the 28K protein of Ad2+ND1: whereas the 28K protein of Ad2+ND1 interacts stably with Nonidet P-40-soluble constituents of the plasma membrane, the 42K and 56K proteins of Ad2+ND2 are tightly bound to the Nonidet P-40-insoluble plasma membrane lamina. Thus, an amino acid sequence in the amino-terminal region of the 28K protein confers membrane affinity to these proteins, whereas a region between the amino-terminal end of the 42K protein of Ad2+ND2 and the amino-terminal end of the 28K protein of Ad2+ND1 contains a reactive site for fatty acid acylation. This posttranslational modification correlates with the stable association of the 42K and 56K proteins with the plasma membrane lamina. We suggest that the same sequences also mediate the proper plasma membrane association of large T-antigen in simian virus 40-transformed cells.

scholarly journals A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Yu Yan ◽  
Ying He ◽  
Bertrand Boson ◽  
Xuesong Wang ◽  
François-Loïc Cosset ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Virus Assembly ◽  
Membrane Association ◽  
Helicase Domain ◽  
Genome Replication ◽  
Virion Assembly ◽  
Adaptive Mutations ◽  
Core Proteins ◽  
Ns3 Protein

ABSTRACT The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α0), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α0, which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix α0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly.

Membrane interactions of simian virus 40 large T-antigen: influence of protein sequences and fatty acid acylation

1984 ◽  
Vol 4 (8) ◽  
pp. 1542-1550
Author(s):  
U Klockmann ◽  
M Staufenbiel ◽  
W Deppert
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Protein Sequences ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Membrane Association ◽  
Large T Antigen ◽  
Amino Terminal

To sort out possible influences of protein sequences and fatty acid acylation on the plasma membrane association of simian virus 40 large T-antigen, we have analyzed the membrane interactions of carboxy-terminal fragments of large T-antigen, encoded by the adenovirus type 2 (Ad2+)-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2. The 28,000 (28K)-molecular-weight protein of Ad2+ND1 as well as the 42K and 56K proteins of Ad2+ND2 associate preferentially with membranous structures and were found in association with the membrane system of the endoplasmic reticulum and with plasma membranes. Neither the endoplasmic reticulum membrane- nor the plasma membrane-associated 28K protein of Ad2+ND1 is fatty acid acylated. We, therefore, conclude that fatty acid acylation is not necessary for membrane association of this protein and suggest that an amino acid sequence in this protein is responsible for its membrane interaction. In contrast, the 42K and 56K proteins of Ad2+ND2 in plasma membrane fractions contain fatty acid. However, the interaction of these proteins with the plasma membrane differs from that of the 28K protein of Ad2+ND1: whereas the 28K protein of Ad2+ND1 interacts stably with Nonidet P-40-soluble constituents of the plasma membrane, the 42K and 56K proteins of Ad2+ND2 are tightly bound to the Nonidet P-40-insoluble plasma membrane lamina. Thus, an amino acid sequence in the amino-terminal region of the 28K protein confers membrane affinity to these proteins, whereas a region between the amino-terminal end of the 42K protein of Ad2+ND2 and the amino-terminal end of the 28K protein of Ad2+ND1 contains a reactive site for fatty acid acylation. This posttranslational modification correlates with the stable association of the 42K and 56K proteins with the plasma membrane lamina. We suggest that the same sequences also mediate the proper plasma membrane association of large T-antigen in simian virus 40-transformed cells.

scholarly journals Retrieval-independent localization of lysyl hydroxylase in the endoplasmic reticulum via a peptide fold in its iron-binding domain

2003 ◽  
Vol 370 (3) ◽  
pp. 913-920 ◽  
Author(s):  
Marko SUOKAS ◽  
Outi LAMPELA ◽  
André H. JUFFER ◽  
Raili MYLLYLÄ ◽  
Sakari KELLOKUMPU
Keyword(s):  
Endoplasmic Reticulum ◽  
Structure Prediction ◽  
Retention Mechanism ◽  
Binding Domain ◽  
Peptide Sequence ◽  
Iron Binding ◽  
Reporter Protein ◽  
Independent Manner ◽  
Lysyl Hydroxylase ◽  
C Terminus

Lysyl hydroxylase (LH) is a peripheral membrane protein in the lumen of the endoplasmic reticulum (ER) that catalyses hydroxylation of lysine residues in collagenous sequences. Previously, we have mapped its primary ER localization motif within a 40-amino acid segment at its C-terminus. Here, we have characterized this localization mechanism in more detail, and our results indicate that this segment confers ER residency in a KDEL-receptor-independent manner, and without any apparent recycling of the enzyme between the Golgi apparatus and the ER. In addition, we show that a rather long peptide region, rather than a specific peptide sequence per se, is required for efficient retention of a reporter protein in the ER. Accordingly, the minimal retention motif was found to require the last 32 C-terminal amino acids, and sequential substitution of all five charged residues within this critical segment interfered only marginally with the retention or association of the enzyme with the ER membranes. Moreover, our fold-recognition and structure-prediction analyses suggested that this critical peptide segment forms an extended loop within LH's iron-binding domain, and that this loop is exposed and readily accessible for binding. Collectively, our results define a novel retrieval-independent retention mechanism in the ER.

scholarly journals The G protein–coupled receptor GPR31 promotes membrane association of KRAS

2017 ◽  
Vol 216 (8) ◽  
pp. 2329-2338 ◽  
Author(s):  
Nicole Fehrenbacher ◽  
Israel Tojal da Silva ◽  
Craig Ramirez ◽  
Yong Zhou ◽  
Kwang-Jin Cho ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Tumor Cells ◽  
G Protein ◽  
Secretory Pathway ◽  
Membrane Association ◽  
G Protein Coupled Receptor ◽  
Genome Wide ◽  
A Genome ◽  
G Protein Coupled

The product of the KRAS oncogene, KRAS4B, promotes tumor growth when associated with the plasma membrane (PM). PM association is mediated, in part, by farnesylation of KRAS4B, but trafficking of nascent KRAS4B to the PM is incompletely understood. We performed a genome-wide screen to identify genes required for KRAS4B membrane association and identified a G protein–coupled receptor, GPR31. GPR31 associated with KRAS4B on cellular membranes in a farnesylation-dependent fashion, and retention of GPR31 on the endoplasmic reticulum inhibited delivery of KRAS4B to the PM. Silencing of GPR31 expression partially mislocalized KRAS4B, slowed the growth of KRAS-dependent tumor cells, and blocked KRAS-stimulated macropinocytosis. Our data suggest that GPR31 acts as a secretory pathway chaperone for KRAS4B.

Sign in / Sign up

Export Citation Format

Share Document