Impaired autoproteolytic cleavage of mCLCA6, a murine integral membrane protein expressed in enterocytes, leads to cleavage at the plasma membrane instead of the endoplasmic reticulum

Ras oncogene proteins are plasma membrane-associated signal transducers that are found in all eukaryotes. Posttranslational addition of lipid to a carboxyl-terminal CaaXbox (where “C” represents a cysteine, “a” is generally an aliphatic residue, andXcan be any amino acid) is required to target Ras proteins to the cytosolic surface of the plasma membrane. The pathway by which Ras translocates from the endoplasmic reticulum to the plasma membrane is currently not clear. We have performed a genetic screen to identify components of the Ras plasma membrane localization pathway. Mutations in two genes,ERF2andERF4/SHR5, have been shown to affect the palmitoylation and subcellular localization of Ras proteins. In this report, we show that Erf4p is localized on the endoplasmic reticulum as a peripheral membrane protein in a complex with Erf2p, an integral membrane protein that was identified from the same genetic screen. Erf2p has been shown to be required for the plasma membrane localization of GFP-Ras2p via a pathway distinct from the classical secretory pathway (X. Dong and R. J. Deschenes, manuscript in preparation). We show here that Erf4p, like Erf2p, is involved in the plasma membrane localization of Ras2p. Erf2p and Erf4p represent components of a previously uncharacterized subcellular transport pathway involved in the plasma membrane targeting of Ras proteins.

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Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm

Journal of Cell Science ◽

10.1242/jcs.111.10.1405 ◽

1998 ◽

Vol 111 (10) ◽

pp. 1405-1418 ◽

Cited By ~ 1

Author(s):

V. Traverso ◽

J.F. Morris ◽

R.J. Flower ◽

J. Buckingham

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Lung Adenocarcinoma ◽

Western Blotting ◽

Signal Sequence ◽

Membrane Surface ◽

Integral Membrane Protein ◽

Subcellular Fractionation ◽

Cell Fractionation ◽

Electron Microscopic

Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.

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Bos1p, an integral membrane protein of the endoplasmic reticulum to Golgi transport vesicles, is required for their competence

Trends in Cell Biology ◽

10.1016/0962-8924(93)90047-5 ◽

1993 ◽

Vol 3 (8) ◽

pp. 256

Author(s):

J.P. Lian ◽

S. Ferro-Novick

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Integral Membrane Protein ◽

Golgi Transport ◽

Transport Vesicles

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Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.3.c982 ◽

2001 ◽

Vol 281 (3) ◽

pp. C982-C992 ◽

Cited By ~ 41

Author(s):

Craig Gatto ◽

Scott M. McLoud ◽

Jack H. Kaplan

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Protein ◽

Atpase Activity ◽

Insect Cells ◽

Expression System ◽

Β Subunit ◽

Α Subunit ◽

Enzyme Preparations ◽

High Five Cells

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.

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Analysis of the Antimalarial Drug Resistance Protein Pfcrt Expressed in Yeast

Journal of Biological Chemistry ◽

10.1074/jbc.m204005200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49767-49775 ◽

Cited By ~ 54

Author(s):

Hanbang Zhang ◽

Ellen M. Howard ◽

Paul D. Roepe

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Integral Membrane Protein ◽

Chloroquine Resistance ◽

Malarial Parasite ◽

Plasma Membrane Vesicle ◽

Wild Type ◽

Stem Loop

Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) inPlasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000)Mol. Cell6, 861–871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane ofSaccharomyces cerevisiaeandPichia pastorisyeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild typeversusCQR-conferring mutant Pfcrt function, which may include effects on H+transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000)Mol. Biochem. Parasitol.110, 107–124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of importantP. falciparumtransport proteins and membrane associated vaccine candidates.

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Cell surface recycling in yeast: mechanisms and machineries

Biochemical Society Transactions ◽

10.1042/bst20150263 ◽

2016 ◽

Vol 44 (2) ◽

pp. 474-478 ◽

Cited By ~ 9

Author(s):

Chris MacDonald ◽

Robert C. Piper

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Membrane Protein ◽

Cell Surface ◽

Mammalian Cells ◽

Genetic System ◽

Integral Membrane Protein ◽

Protein Activity ◽

Yeast Saccharomyces Cerevisiae ◽

Central Process

Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeast Saccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.

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Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1

Biochemical Journal ◽

10.1042/bj20030884 ◽

2004 ◽

Vol 379 (1) ◽

pp. 31-38 ◽

Cited By ~ 63

Author(s):

Emily R. SLEPKOV ◽

Signy CHOW ◽

M. Joanne LEMIEUX ◽

Larry FLIEGEL

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Mammalian Cells ◽

Integral Membrane Protein ◽

Amide Hydrogen ◽

Wild Type ◽

Transmembrane Segment ◽

Mutant Proteins ◽

Transmembrane Segments ◽

Induced Changes

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167→Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE.

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Infectious Bronchitis Virus 3a Protein Localizes to a Novel Domain of the Smooth Endoplasmic Reticulum

Journal of Virology ◽

10.1128/jvi.79.10.6142-6151.2005 ◽

2005 ◽

Vol 79 (10) ◽

pp. 6142-6151 ◽

Cited By ~ 10

Author(s):

Amanda R. Pendleton ◽

Carolyn E. Machamer

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Infectious Bronchitis Virus ◽

Smooth Endoplasmic Reticulum ◽

Integral Membrane Protein ◽

Open Reading Frames ◽

Infectious Bronchitis ◽

Infected Cells ◽

Group 3 ◽

Small Open Reading Frames

ABSTRACT All coronaviruses possess small open reading frames (ORFs) between structural genes that have been hypothesized to play important roles in pathogenesis. Infectious bronchitis virus (IBV) ORF 3a is one such gene. It is highly conserved among group 3 coronaviruses, suggesting that it has an important function in infection. IBV 3a protein is expressed in infected cells but is not detected in virions. Sequence analysis predicted that IBV 3a was a membrane protein; however, only a fraction behaved like an integral membrane protein. Microscopy and immunoprecipitation studies demonstrated that IBV 3a localized to the cytoplasm in a diffuse pattern as well as in sharp puncta in both infected and transfected cells. These puncta did not overlap cellular organelles or other punctate structures. Confocal microscopy demonstrated that IBV 3a puncta lined up along smooth endoplasmic reticulum (ER) tubules and, in a significant number of instances, were partially surrounded by these tubules. Our results suggest that IBV 3a is partially targeted to a novel domain of the smooth ER.

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Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies.

The Journal of Cell Biology ◽

10.1083/jcb.98.6.2142 ◽

1984 ◽

Vol 98 (6) ◽

pp. 2142-2147 ◽

Cited By ~ 109

Author(s):

P Quinn ◽

G Griffiths ◽

G Warren

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Proteins ◽

Membrane Protein ◽

Semliki Forest Virus ◽

Companion Paper ◽

Intracellular Membranes ◽

Surface Areas ◽

Quantitative Immunoblotting ◽

Total Membrane

Using two independent methods, incorporation of radioactive amino-acid and quantitative immunoblotting, we have determined that the rate of synthesis of each of the Semliki Forest virus (SFV) proteins in infected baby hamster kidney (BHK) cells is 1.2 X 10(5) copies/cell/min. Given the absolute surface areas of the endoplasmic reticulum and Golgi complex presented in the companion paper (Griffiths, G., G. Warren, P. Quinn , O. Mathieu - Costello , and A. Hoppeler , 1984, J. Cell Biol. 98:2133-2141), and the approximate time spent in these organelles during their passage to the plasma membrane (Green J., G. Griffiths, D. Louvard , P. Quinn , and G. Warren 1981, J. Mol. Biol. 152:663-698), the mean density of each viral protein in these organelles can be calculated to be 90 and 750 molecules/micron 2 membrane, respectively. In contrast, we have determined that the density of total endogenous integral membrane proteins in these organelles is approximately 30,000 molecules/micron 2 so that the spike proteins constitute only 0.28 and 2.3% of total membrane protein in the endoplasmic reticulum and Golgi, respectively. Quantitative immunoblotting was used to give direct estimates of the concentrations of one of the viral membrane protein precursors (E1) in subcellular fractions; these agreed closely with the calculated values. The data are discussed with respect to the sorting of transported proteins from those endogenous to the intracellular membranes.

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