The Golgi apparatus and membrane trafficking in green algae.
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Biological Transport↗
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Results for “Membrane trafficking”
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Disruptions in intracellular membrane trafficking and structure preclude the glucocorticoid-dependent maturation of mouse mammary tumor virus proteins in rat hepatoma cells.
We have previously shown that glucocorticoids regulate the trafficking and processing of mouse mammary tumor virus (MMTV) proteins in viral-infected M1.54 rat hepatoma cells. To examine the role of intracellular membrane integrity on MMTV protein maturation, brefeldin A (BFA) was utilized to disrupt membrane flow between the endoplasmic reticulum and Golgi. Immunoprecipitation and immunofluorescence microscopy revealed that in the presence of dexamethasone, BFA inhibited the proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins. Glycosidase digestion and inhibitors of protein glycosylation confirmed that the observed differences in apparent sizes of MMTV glycoprotein products are due to BFA-induced changes in oligosaccharide processing. BFA treatment inhibited the proteolytic processing of the MMTV phosphoprotein precursor, which normally associates with the cytoplasmic face of intracellular membranes. Similarities in salt extraction efficiency revealed that BFA did not affect the membrane affinity of the uncleaved phosphorylated precursor. In a complementary approach, proteolytic processing of the phosphorylated polyprotein did not occur in glucocorticoid-treated HTC cells transfected with a mutant MMTV provirus encoding a normal phosphorylated precursor, but which express a truncated MMTV glycoprotein missing its transmembrane domain and cytoplasmic tail. These results suggest that the MMTV glycoproteins and phosphoproteins may interact at a late step in the transport pathway in a manner required for their mutual processing in response to glucocorticoids and establishes the importance of functional interactions with intracellular membranes for maturation of the cytoplasmic MMTV phosphoproteins.
Animals↗
Membrane trafficking in neurons.
Neurons possess an unusually extensive Golgi apparatus and exhibit a variety of active endocytic-like processes. The Golgi apparatus and the endocytic phenomena both contribute, probably in multiple overlapping ways, to the genesis and fate of the membrane systems in axons and terminals.
Animals↗
Selective membrane protein trafficking: vectorial flow and filter.
Membrane proteins trafficking along cellular pathways encounter molecular filters. These filters can introduce them to new pathways and thus direct them towards new destinations. Many proteins carry molecular signals within their cytoplasmic domains that allow them to be selected by the filters. The detailed characterization of these signals is providing new insights into the pathways themselves and indicating the locations of other, as yet unidentified filters.
Amino Acid Sequence↗
Effects of the expression of mammalian annexins in yeast secretory mutants.
The hypothesis that calcium-dependent membrane-binding proteins of the annexin family can influence intracellular membrane trafficking was tested by expressing five mammalian annexins in wild-type yeast cells (Saccharomyces cerevisiae) and in 13 yeast secretory (sec) mutants. Expression of human synexin (annexin VII) inhibited the growth of sec2, sec4 and sec15 mutants at a semi-permissive temperature. These three sec mutants are defective in the final step in the secretory pathway, the process of exocytosis. The inhibition of growth correlated with reduced viability and increased accumulation of internal invertase in these mutants when expressing synexin. Bovine endonexin (annexin IV) partially suppressed the growth defect of a sec2 mutant incubated at a semi-permissive temperature. Human synexin, human lipocortin (annexin I), and murine p68 (annexin VI) reduced the lag time associated with adaptation of sec2 mutants to galactose-containing medium. These interactions suggest that the annexins may influence specific steps in membrane trafficking associated with cell growth, secretion and plasma membrane remodelling.
Annexins↗
Regulated membrane vesicle trafficking: a defect in cystic fibrosis corrected by gene transfer.
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Cell Line↗
Acyl-trafficking in membrane phospholipid fatty acid turnover: the transfer of fatty acid from the acyl-L-carnitine pool to membrane phospholipids in intact human erythrocytes.
In this work we have investigated the transfer of radioactive palmitic acid between membrane phospholipids and acyl-L-carnitines in intact human erythrocytes. During the incubation period of labeled erythrocyte in non-defatted bovine serum albumin, radioactivity in phosphatidylcholine and phosphatidylethanolamine increased. On the contrary, a decrease of radioactivity in erythrocyte palmitoyl-L-carnitine was observed. 2-Tetradecylglycidic acid, an irreversible erythrocyte carnitine palmitoyltransferase inhibitor, abolished any radioactivity changes in both phospholipids and palmitoyl-L-carnitine. Similar findings were obtained by using erythrocytes labeled with radioactive oleic acid. Our data suggest that in human erythrocytes a carnitine palmitoyltransferase-catalyzed acyl transfer from acyl-L-carnitine to phospholipids, rather than a previously described fatty acid transfer from phosphatidylcholine to phosphatidylethanolamine, is operative.
Carnitine O-Palmitoyltransferase↗
Use of the D4H Probe to Track Sterols in Yeast.
Cholesterol is a fundamental component of cellular membranes, and its organization, distribution, and recycling are tightly regulated. Cholesterol can form, together with other lipids and proteins, membrane nanodomains, which play important roles in membrane trafficking, the spatiotemporal organization of signal transduction, or the modulation of plasma membrane transporters, among others. Not surprisingly then, the misregulation of cholesterol biosynthetic and transport pathways has been related to numerous diseases, including neurodegenerative and metabolic disorders. Here, we focus on the cholesterol-binding domain 4 (D4) of perfringolysin O (PFO, theta toxin) and its use as a probe to define the dynamics and subcellular localization of yeast sterols using time-lapse live-cell fluorescence microscopy. In combination with drugs that acutely interfere with sterol synthesis, such as terbinafine, the probe can also be used to monitor in real-time the extraction of sterols from specialized endoplasmic reticulum subdomains named ERSES (endoplasmic reticulum sterol exit sites) by the OSBP-related protein Osh2.
Saccharomyces cerevisiae↗
Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins.
The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference to the fractions of these proteins which are membrane-bound. In addition to EGTA-extractable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilized with detergents (Triton X-100 or Triton X-114). A strong base like Na2CO3, which is usually effective in extracting membrane proteins, only partially solubilizes the membrane-bound, EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble annexins partition in the aqueous phase, the remaining fractions being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as yet unknown mechanism, following Ca(2+)-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of these proteins remain bound to membranes in a Ca(2+)-independent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.
Animals↗
Identification of rab2 as a tubulovesicle-membrane-associated protein in rabbit gastric parietal cells.
Rab proteins, which are ras-like low-molecular-mass GTP-binding proteins, are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. Previously, we reported a 23 kDa tubulovesicle-associated GTP-binding protein in rabbit gastric parietal cells [Basson, Goldenring, Tang, Lewis, Padfield, Jamieson & Modlin (1991) Biochem. J. 279, 43-48]. The major component of the 23 kDa protein is now identified as rab2. Rab2 was co-localized in tubulovesicle membranes from parietal cells. Consistent with GTP-binding activity (as documented before), upon maximal stimulation of parietal cells, rab2 immunoreactivity was redistributed from a 50,000 g to a 4000 g subcellular membrane fraction. The tubulovesicle-associated rab2 behaved as an integral membrane protein, since both 0.5 M-NaCl and 0.1 M-carbonate extraction failed to remove the protein from the tubulovesicle membrane. Utilizing a PCR the rab2 cDNA sequence from rabbit parietal cells was obtained, and it showed only one amino acid difference compared with the human sequence. The results of the present study provide strong evidence that parietal cells possess a rab2 protein which is tightly associated with tubulovesicle membranes.
Adenosine Triphosphatases↗
Membrane fusion.
Common themes are emerging from the study of viral, cell-cell, intracellular, and liposome fusion. Viral and cellular membrane fusion events are mediated by fusion proteins or fusion machines. Viral fusion proteins share important characteristics, notably a fusion peptide within a transmembrane-anchored polypeptide chain. At least one protein involved in a cell-cell fusion reaction resembles viral fusion proteins. Components of intracellular fusion machines are utilized in multiple membrane trafficking events and are conserved through evolution. Fusion pores develop during and intracellular fusion events suggesting similar mechanisms for many, if not all, fusion events.
Biological Transport↗
FTO promotes weight gain via altering Kif1a splicing and axonal vesicle trafficking in AgRP neurons.
N6-methyladenosine (m6A) is an abundant chemical RNA modification involved in the regulation of many biological processes. The m6A demethylase FTO (fat mass and obesity-associated protein) is known to affect body weight, but its systemic context and underlying mechanisms remain unclear. Here, we found that mice lacking or overexpressing Fto in agouti-related peptide-expressing (AgRP) neurons in the hypothalamus exhibited decreased and increased body weight, respectively. FTO demethylated m6A on mRNAs for proteins associated with membrane trafficking and alternative splicing in AgRP neurons. Downstream, FTO-modulated alternative splicing of the axonal motor protein Kif1a affected its hinge region, which is relevant to the structure and function of KIF1A. Notably, Kif1a knockdown in AgRP neurons suppressed the weight gain of mice overexpressing Fto. In addition, FTO increased the trafficking and secretion of dense-core vesicles containing neuropeptides NPY and AgRP from AgRP neurons. Collectively, these results reveal a novel regulatory FTO-KIF1A axis in the brain affecting appetite-stimulating AgRP neurons and systemic energy homeostasis, via FTO regulation of the epitranscriptome of AgRP neurons.
Animals↗
Immunocytochemical localization of annexin V (CaBP33), a Ca(2+)-dependent phospholipid- and membrane-binding protein, in the rat nervous system and skeletal muscles and in the porcine heart.
We investigated the ultrastructural localization of annexin V a Ca(2+)-dependent phospholipid- and membrane-binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca(2+)-dependent binding of the protein to brain and heart membranes, and on existence of both EGTA- and Triton X-100-extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells.
Animals↗
Disruption of Polycystin Ciliary Localization and Channel Function by Autosomal Dominant Polycystic Kidney Disease-Causing Polycystin-1 Variants.
KEY POINTS: We developed assays to measure genetic variant effects on polycystin-1, the protein mutated in most autosomal dominant polycystic kidney disease. All tested pathogenic variants disrupted either polycystin-1 ciliary trafficking or channel function. Trafficking and channel function of some pathogenic variants was restored by low temperature culture to promote polycystin folding. BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the leading monogenic cause of kidney failure and affects millions of people worldwide. Despite the prevalence of ADPKD, limited mechanistic understanding has hindered therapeutic development. Most ADPKD is caused by loss-of-function variants in polycystin-1 (PC1). METHODS: We developed assays that quantify the effect of nontruncating variants on PC1 ciliary localization, membrane trafficking, and polycystin channel function. RESULTS: We evaluated 29 nontruncating variants in PC1 and found that pathogenic variants disrupt two molecular phenotypes: ( 1 ) localization of PC1 at the primary cilium or ( 2 ) polycystin ion channel activity. Ciliary localization of a subset of polycystin variants was restored when cells were cultured at low temperature. A subset of variants with localization restored by low temperature formed functional channels. CONCLUSIONS: This study demonstrated that disruptions in polycystin ciliary trafficking and channel function are common causes of ADPKD. Defects in ciliary trafficking and channel function can be rescued for a subset of pathogenic variants, establishing a foundation for polycystin-targeted therapies in ADPKD.
Polycystic Kidney, Autosomal Dominant↗
Small GTP-binding proteins in parietal cells: candidate modulators of parietal cell membrane dynamics.
The stimulated fusion of intracellular H/K-ATPase-containing tubulovesicles with a target canalicular membrane surface is central to the process of acid secretion. A super-family of small GTP-binding proteins (smGTPBPs) has been implicated in many aspects of intracellular dynamics and vesicle membrane trafficking. We have investigated the presence of smGTPBPs in isolated rabbit parietal cells. Parietal cells possess a number of smGTPBP species with molecular masses of 18-28 kDa. One 23 kDa smGTPBP has been localized to tubulovesicles and identified immunochemically as rab2. Rab2 redistributes during stimulation in concert with the movement of the H/K-ATPase. The results demonstrate that specific smGTPBPs are associated with the parietal cell secretory apparatus. Small GTP-binding proteins are important candidate regulators of parietal secretory membrane dynamics.
Animals↗
Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles.
The F-antigen is a prominent liver protein which has been extensively used in studies on natural and induced immunological tolerance. However, its intracellular localization and biological function have remained elusive. It has generally been assumed that the F-antigen is confined phylogenetically to vertebrates. Now we have cloned and characterized a gene from the ciliated protozoan Tetrahymena thermophila encoding a protein which clearly is homologous with the rat F-antigen. The coding region of the Tetrahymena F-antigen (TF-ag) gene specifies a 46,051 M(r) protein and is interrupted by three introns. In accordance with the predicted molecular mass of the TF-ag protein, antibodies raised against a cro-lacZ'-TF-ag fusion protein specifically recognized a 45,000 M(r) protein in Western blots of total T. thermophila protein. Immunoelectron microscopy demonstrated that the TF-ag is associated with membranes of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking. Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded. The abundance of the TF-ag protein, however, declined only moderately during prolonged periods of starvation demonstrating that extensive release of the TF-ag did not take place. In combination these results suggest that the TF-ag protein is a recycled constituent of the intracellular membrane network in T. thermophila.
Amino Acid Sequence↗
Association of a 19- and a 21-kDa GTP-binding protein to pancreatic microsomal vesicles is regulated by the intravesicular pH established by a vacuolar-type H(+)-ATPase.
Evidence suggests that certain ras-related small molecular weight GTP-binding proteins (smg-proteins) are involved in intracellular membrane trafficking and vesicle fusion. We have previously shown that intravesicular acidification due to a vacuolar-type H(+)-ATPase, which is Cl- dependent and highly sensitive to the specific inhibitor bafilomycin, enhances GTP-induced fusion of pancreatic microsomal vesicles (Hampe, W., Zimmermann, P., Schulz, I. 1990. FEBS Lett. 271:62-66). This process may involve function of smg-proteins. The present study shows that MgATP (2 mM), but neither MgATP gamma S nor ATP in the absence of Mg2+, increases association of 19- and 21-kDa smg-proteins to the vesicle membrane as monitored by their [ alpha-32P]GTP binding. The affinity of smg-proteins for [ alpha-32P]GTP was not altered by MgATP. Bafilomycin B1 (10(-8) M), the protonophore CCCP (10(-5) M), and replacement of Cl- in the incubation buffer by CH3COO- or NO3- resulted in an almost complete inhibition of the MgATP-dependent association of the 19- and 21-kDa smg-proteins to the vesicle membranes. Furthermore, the MgATP effect on both smg-proteins was found to be due to the intravesicular pH and not to the H+ gradient over the vesicle membrane. We conclude that association of a 19-kDa (immunologically identified as the ADP-ribosylation factor, arf) and a yet unidentified 21-kDa GTP-binding protein to vesicle membranes is regulated by the intravesicular pH established by a vacuolar-type H(+)-ATPase.
Adenosine Triphosphate↗
Phosphorylation of annexin II tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein.
Annexin II tetramer (A-IIt) is a member of the annexin family of Ca2+ and phospholipid-binding proteins. The ability of this protein to aggregate both phospholipid vesicles and chromaffin granules has suggested a role for the protein in membrane trafficking events such as exocytosis. A-IIt is also a major intracellular substrate of both pp60src and protein kinase C; however, the effect of phosphorylation on the activity of this protein is unknown. In the current report we have examined the effect of phosphorylation on the lipid vesicle aggregation activity of the protein. Protein kinase C catalyzed the incorporation of 2.1 +/- 0.8 mol of phosphate/mol of A-IIt. Phosphorylation of A-IIt caused a dramatic decrease in the rate and extent of lipid vesicle aggregation without significantly effecting Ca(2+)-dependent lipid binding by the phosphorylated protein. Phosphorylation of A-IIt increased the A50%(Ca2+) of lipid vesicle aggregation from 0.18 microM to 0.65 mM. Activation of A-IIt phosphorylation, concomitant with activation of lipid vesicle aggregation, inhibited both the rate and extent of lipid vesicle aggregation but did not cause disassembly of the aggregated lipid vesicles. These results suggest that protein kinase C-dependent phosphorylation of A-IIt blocks the ability of the protein to aggregate phospholipid vesicles without affecting the lipid vesicle binding properties of the protein.
Amino Acids↗