scholarly journals Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes

2020 ◽  
Vol 31 (9) ◽  
pp. 2044-2064 ◽  
Author(s):  
Suzie J. Scales ◽  
Nidhi Gupta ◽  
Ann M. De Mazière ◽  
George Posthuma ◽  
Cecilia P. Chiu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
High Frequency ◽  
Lipid Droplets ◽  
Human Kidney ◽  
Cytoplasmic Face ◽  
Large Panel ◽  
Surface Localization ◽  
Apolipoprotein L1

BackgroundAPOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs.MethodsImmunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies.ResultsBoth endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants.ConclusionsAPOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.

scholarly journals C. elegansparaoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins

2016 ◽  
Author(s):  
Yushu Chen ◽  
Shashank Bharill ◽  
Zeynep Altun ◽  
Robert O'Hagan ◽  
Brian Coblitz ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Cell Surface ◽  
Functional Expression ◽  
Similar Protein ◽  
Touch Sensitivity ◽  
Additional Protein

Caenorhabditis eleganssenses gentle touch via a mechanotransduction channel formed from the DEG/ENaC proteins MEC-4 and MEC-10. An additional protein, the paraoxonase-like protein MEC-6, is essential for transduction, and previous work suggested that MEC-6 was part of the transduction complex. We found that MEC-6 and a similar protein, POML-1, reside primarily in the endoplasmic reticulum and do not colocalize with MEC-4 on the plasma membrane in vivo. As with MEC-6, POML-1 is needed for touch sensitivity, for the neurodegeneration caused by themec-4(d)mutation, and for the expression and distribution of MEC-4 in vivo. Both proteins are likely needed for the proper folding or assembly of MEC-4 channels in vivo as measured by FRET. MEC-6 detectably increases the rate of MEC-4 accumulation on theXenopusoocyte plasma membrane. These results suggest that MEC-6 and POML-1 interact with MEC-4 to facilitate expression and localization of MEC-4 on the cell surface. Thus, MEC-6 and POML-1 act more like chaperones for MEC-4 than channel components.

Studies of the endoplasmic reticulum and plasma membrane-bound ribosomes in erythropoietic cells

1978 ◽  
Vol 31 (1) ◽  
pp. 165-178
Author(s):  
J.A. Grasso ◽  
A.L. Sullivan ◽  
S.C. Chan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Exact Role ◽  
Whole Cells ◽  
Cytoplasmic Face ◽  
Close Proximity ◽  
Haem Biosynthesis ◽  
Hypotonic Buffer ◽  
Membrane Bound ◽  
Hypotonic Lysis

Erythropoietic cells of 5 species, including man, contain endoplasmic reticulum present as individual cisternae or tubules scattered throughout the cytoplasm of all stages except mature RBCs. The endoplasmic reticulum is mainly agranular but occurs frequently as a variant of granular ER which is characterized by an asymmetrical and irregular distribution of ribosomes along one cytoplasmic face. In most cells, the endoplasmic reticulum occurs in close proximity to mitochondria or the plasma membrane, suggesting that the organelle may be involved in functions related to these structures, e.g. haem biosynthesis. Endoplasmic reticulum is more abundant in early than in late erythroid cells. Its exact role in RBC development is unclear. Since endoplasmic reticulum could account for ‘plasma membrane-bound ribosomes’ reported in lysed reticulocytes, studies were performed which ruled out this possibility and which suggested that such ribosomes were an artifact of the lysing conditions. Hypotonic lysis in less than 20 vol. of magnesium-containing buffers yielded ghosts variably contaminated by ribosomes and other structures. Lysis of reticulocytes in 20–30 vol. of magnesium-free buffer or homogenization of whole cells or crude membrane fractions in hypotonic buffer removed virtually all contaminating ribosomes from the purified membrane fraction.

scholarly journals RAB6 and microtubules restrict protein secretion to focal adhesions

2019 ◽  
Vol 218 (7) ◽  
pp. 2215-2231 ◽  
Author(s):  
Lou Fourriere ◽  
Amal Kasri ◽  
Nelly Gareil ◽  
Sabine Bardin ◽  
Hugo Bousquet ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Secretion ◽  
Golgi Complex ◽  
Essential Role ◽  
Focal Adhesions ◽  
Secreted Proteins ◽  
Cell Compartments ◽  
Secretion Assay

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

scholarly journals A variant of the pyroantimonate technique suitable for localization of calcium in ovarian tissue.

1979 ◽  
Vol 27 (6) ◽  
pp. 1017-1028 ◽  
Author(s):  
B S Weakley
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Lipid Droplets ◽  
Microprobe Analysis ◽  
Smooth Endoplasmic Reticulum ◽  
Ovarian Tissue ◽  
Follicle Cells ◽  
Fixation Time ◽  
X Ray ◽  
Cytoplasmic Processes

Osmium-pyroantimonate solutions for the precipitation of cations are unsuitable for use with delicate mammalian oocytes. A variant of the pyroantimonate technique employing a mixture of pyroantimonate and glutaraldehyde has been found to give successful and repeatable results if a fixation time of 4 hr is used. Calcium-containing antimonate precipitates were localized principally in nuclei, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, and cytoplasmic processes of both oocytes and follicle cells, and along the plasma membrane in small oocytes. Deposits were also concentrated around the periphery of lipid droplets in the follicle cells. The presence of calcium in the precipitates was confirmed by x-ray microprobe analysis.

scholarly journals Subcellular distribution of ezrin/radixin/moesin and their roles in the cell surface localization and transport function of P-glycoprotein in human colon adenocarcinoma LS180 cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0250889
Author(s):  
Takuro Kobori ◽  
Mayuka Tameishi ◽  
Chihiro Tanaka ◽  
Yoko Urashima ◽  
Tokio Obata
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cancer Cells ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Membrane Localization ◽  
Transport Function ◽  
Erm Proteins ◽  
Plasma Membrane Localization ◽  
Surface Localization

The ezrin/radixin/moesin (ERM) family proteins act as linkers between the actin cytoskeleton and P-glycoprotein (P-gp) and regulate the plasma membrane localization and functionality of the latter in various cancer cells. Notably, P-gp overexpression in the plasma membrane of cancer cells is a principal factor responsible for multidrug resistance and drug-induced mutagenesis. However, it remains unknown whether the ERM proteins contribute to the plasma membrane localization and transport function of P-gp in human colorectal cancer cells in which the subcellular localization of ERM has yet to be determined. This study aimed to determine the gene expression patterns and subcellular localization of ERM and P-gp and investigate the role of ERM proteins in the plasma membrane localization and transport function of P-gp using the human colon adenocarcinoma cell line LS180. Using real-time reverse transcription polymerase chain reaction and immunofluorescence analyses, we showed higher levels of ezrin and moesin mRNAs than those of radixin mRNA in these cells and preferential distribution of all three ERM proteins on the plasma membrane. The ERM proteins were highly colocalized with P-gp. Additionally, we show that the knockdown of ezrin, but not of radixin and moesin, by RNA interference significantly decreased the cell surface expression of P-gp in LS180 cells without affecting the mRNA expression of P-gp. Furthermore, gene silencing of ezrin substantially increased the intracellular accumulation of rhodamine123, a typical P-gp substrate, with no alterations in the plasma membrane permeability of Evans blue, a passive transport marker. In conclusion, ezrin may primarily regulate the cell surface localization and transport function of P-gp as a scaffold protein without influencing the transcriptional activity of P-gp in LS180 cells. These findings should be relevant for treating colorectal cancer, which is the second leading cause of cancer-related deaths in males and females combined.

Smooth Muscular Cells in the Mammalian Ovary

1971 ◽  
Vol 29 ◽  
pp. 508-509
Author(s):  
Z. Fumagalli ◽  
P. Motta ◽  
S. Calvieri
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Electron Microscope ◽  
Small Groups ◽  
Cortical Area ◽  
Lipid Droplets ◽  
Corpora Lutea ◽  
Ovarian Stroma ◽  
Theca Interna ◽  
Glycogen Particles

The presence of smooth muscular cells was demonstrated with the electron microscope in different areas of the ovary of cats, mice and rabbits. The myocytes were arranged in fascicles, small groups, or most frequently appeared isolated. They were scattered in the ovarian stroma, related to the interstitial cells, in the periphery of the corpora lutea (rarely between luteal cells) in the middle of the gland. Smooth muscular cells were seldom observed between cells of the theca interna and externa of developing follicles and in the middle of atresic follicles. Some smooth muscular cells were found in the cortical area of the ovaries.Each smooth muscular cell showed typical filaments, free ribosomes, lipid droplets and at times glycogen particles. Mitochondria were vesicular; the (Golgi) vesicular complex was often related to two centrioles (frequently in a process of ciliogenesis). The granular endoplasmic reticulum was moderately developed. The plasma membrane presented invaginations and micropinocytotic vesicles as well as tight junctions between adjacent cells. The nucleus was elongated and its envelope formed wide perinuclear cisternae.

scholarly journals SAT-LB137 Rescue of Misfolded G-Protein Coupled Estrogen Receptor, GPER, from the Endoplasmic Reticulum via Natural and Synthetic Estrogens

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Milad Rouhimoghadam ◽  
Jing Dong ◽  
Peter Thomas ◽  
Edward Joseph Filardo
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Surface Expression ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
17Β Estradiol ◽  
G Protein Coupled

Abstract GPER bears structural and functional characteristics shared by members of the G-protein coupled receptor (GPCR) superfamily, the largest class of cell surface receptors, with more than 800 members encoded in the human genome. GPER is localized predominately in intracellular membranes, in many but not all cell types, and its surface expression is modulated by steroid hormones and during tissue homeostasis. An intracellular staining pattern is not unique among GPCRs, which deploy a diverse array of posttranslational regulatory mechanisms to determine cell surface expression, effectively regulating cognate ligand binding and activity. Here, we show nascent GPER undergoes strict quality control via endoplasmic reticulum associated degradation (ERAD) requiring direct poly-ubiquitinylation of GPER and valosin-containing protein VCP/p97-mediated segregation of misfolded proteins from the ER membrane to the cytoplasm for delivery to the 26S proteasome. Specifically, we find that inhibition of p97 using the pharmacological compound, CB-5083, or by doxycycline-inducible p97 shRNA results in the accumulation of immature glycosylated GPER in the ER. Inhibition of proteasome function facilitates anterograde trafficking with the transport of nonfunctional GPER to the plasma membrane as indicated by no increase in specific estrogen binding using 3H-17β-estradiol in a radioreceptor assay. The forward trafficking of misfolded GPER requires transit through the Golgi as treatment with brefeldin A (BFA) prevents GPER plasma membrane expression. Substitution of all three lysines (K333, K342, and K357) encoded in the cytoplasmic tail of GPER with arginines blunts its polyubiquitinylation and allows GPER to evade degradation by quality control but does not result in increased plasma membrane expression suggesting that additional structural motifs encoded within GPER control its anterograde trafficking. In contrast, functional GPER is recovered at the plasma membrane of human SKBR3 breast cancer cells treated with either 17β-estradiol or the GPER selective antagonist, G15, in the presence of cycloheximide resulting in increased surface GPER. Thus, our findings suggest that estrogens, both natural and synthetic, can function as pharmacochaperones capable of promoting the correct folding of GPER and enhanced expression of functional GPER at the plasma membrane.

scholarly journals A Novel Ras Inhibitor, Eri1, Engages Yeast Ras at the Endoplasmic Reticulum

2003 ◽  
Vol 23 (14) ◽  
pp. 4983-4990 ◽  
Author(s):  
Andrew K. Sobering ◽  
Martin J. Romeo ◽  
Heather A. Vay ◽  
David E. Levin
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Regulatory Function ◽  
Ras Signaling ◽  
Cell Proliferation And Differentiation ◽  
Acid Protein ◽  
Cytoplasmic Face ◽  
Proliferation And Differentiation ◽  
Mutant Forms

ABSTRACT Ras oncoproteins are monomeric GTPases that link signals from the cell surface to pathways that regulate cell proliferation and differentiation. Constitutively active mutant forms of Ras are found in ca. 30% of human tumors. Here we report the isolation of a novel gene from Saccharomyces cerevisiae, designated ERI1 (for endoplasmic reticulum-associated Ras inhibitor 1), which behaves genetically as an inhibitor of Ras signaling. ERI1 encodes a 68-amino-acid protein that associates in vivo with GTP-bound Ras in a manner that requires an intact Ras-effector loop, suggesting that Eri1 competes for the same binding site as Ras target proteins. We show that Eri1 localizes primarily to the membrane of the endoplasmic reticulum (ER), where it engages Ras. The recent demonstration that signaling from mammalian Ras is not restricted to the cell surface but can also proceed from the cytoplasmic face of the ER suggests a regulatory function for Eri1 at that membrane.

scholarly journals The Subcellular Distribution of Calnexin Is Mediated by PACS-2

2008 ◽  
Vol 19 (7) ◽  
pp. 2777-2788 ◽  
Author(s):  
Nathan Myhill ◽  
Emily M. Lynes ◽  
Jalal A. Nanji ◽  
Anastassia D. Blagoveshchenskaya ◽  
Hao Fei ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Kinase Ck2 ◽  
Phosphorylation State ◽  
Calcium Pumps ◽  
Rough Er ◽  
Er Homeostasis ◽  
Kinase Ck2 ◽  
Er Calcium

Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 [Formula: see text] Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.

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