Jacques Paiement

Endoplasmic Reticulum-Mediated Phagocytosis Is a Mechanism of Entry into Macrophages

Phagocytosis is a key aspect of our innate ability to fight infectious diseases. In this study, we have found that fusion of the endoplasmic reticulum (ER) with the macrophage plasmalemma, underneath phagocytic cups, is a source of membrane for phagosome formation in macrophages. Successive waves of ER become associated with maturing phagosomes during phagolysosome biogenesis. Thus, the ER appears to possess unexpectedly pluripotent fusion properties. ER-mediated phagocytosis is regulated in part by phosphatidylinositol 3-kinase and used for the internalization of inert particles and intracellular pathogens, regardless of their final trafficking in the host. In neutrophils, where pathogens are rapidly killed, the ER is not used as a major source of membrane for phagocytosis. We propose that intracellular pathogens have evolved to adapt and exploit ER-mediated phagocytosis to avoid destruction in host cells.

Quantitative Proteomics Analysis of the Secretory Pathway

We report more than 1400 proteins of the secretory-pathway proteome and provide spatial information on the relative presence of each protein in the rough and smooth ER Golgi cisternae and Golgi-derived COPI vesicles. The data support a role for COPI vesicles in recycling and cisternal maturation, showing that Golgi-resident proteins are present at a higher concentration than secretory cargo. Of the 1400 proteins, 345 were identified as previously uncharacterized. Of these, 230 had their subcellular location deduced by proteomics. This study provides a comprehensive catalog of the ER and Golgi proteomes with insight into their identity and function.

Increased Association Between Rough Endoplasmic Reticulum Membranes and Mitochondria in Transgenic Mice That Express P301L Tau

In several neurodegenerative diseases, including Alzheimer disease, the neuronal microtubule-associated protein tau becomes hyperphosphorylated, accumulates in the somatodendritic compartment, and aggregates into insoluble filaments. The consequences of the accumulation of hyperphosphorylated tau in the somatodendritic compartment remain poorly characterized at the early stage of disease before the formation of tau insoluble filaments. We investigated the ultrastructural changes induced by this accumulation in the neuronal soma of motor neurons in asymptomatic JNPL3 mice that overexpress mutant tau, P301L. More numerous contacts between rough endoplasmic reticulum (RER) membranes and mitochondria were observed in JNLP3 mice compared with wild-type mice. This correlated with a preferential increase of the amount of tau at the surface of RER membranes but not at the surface of mitochondria, as revealed by tau immunogold labeling. Using a subcellular fractionation procedure, an increased amount of phosphorylated tau was identified in the rough microsome subfraction, wherein the RER marker, ribophorin, was enriched. A similar increase was noted in the rough microsome subfraction isolated from Alzheimer disease brains. The association of hyperphosphorylated tau with ER membranes was confirmed by double immunogold labeling of the subfraction enriched in ER membranes isolated from Alzheimer disease brains. These results suggest that more contacts between RER membranes and mitochondria resulting from the accumulation of tau at the surface of RER membranes might contribute to tau-induced neurodegeneration.

Topology of molecular machines of the endoplasmic reticulum: a compilation of proteomics and cytological data

The endoplasmic reticulum (ER) is a key organelle of the secretion pathway involved in the synthesis of both proteins and lipids destined for multiple sites within and without the cell. The ER functions to both co- and post-translationally modify newly synthesized proteins and lipids and sort them for housekeeping within the ER and for transport to their sites of function away from the ER. In addition, the ER is involved in the metabolism and degradation of specific xenobiotics and endogenous biosynthetic products. A variety of proteomics studies have been reported on different subcompartments of the ER providing an ER protein dictionary with new data being made available on many protein complexes of relevance to the biology of the ER including the ribosome, the translocon, coatomer proteins, cytoskeletal proteins, folding proteins, the antigen-processing machinery, signaling proteins and proteins involved in membrane traffic. This review examines proteomics and cytological data in support of the presence of specific molecular machines at specific sites or subcompartments of the ER.

Physiological concentrations of GTP stimulate fusion of the endoplasmic reticulum and the nuclear envelope

Incubation of highly purified nuclei with rough microsomes stripped of associated ribosomes and physiological concentrations of guanosine triphosphate (GTP) led to the fusion of outer membranes of nuclei with microsomes to form large irregular membrane extensions. Measurement of membrane profiles in electron micrographs revealed that the outer membranes of nuclei incubated under these conditions increased significantly in length compared with that of outer membranes of unincubated or control incubated nuclei. This morphometric assay for fusion was used to check membrane and tissue specificity. It was found that GTP did not stimulate fusion between other intracellular membranes (e.g. mitochondrial or Golgi) or between such membranes and nuclear envelopes. GTP did, however, stimulate fusion between stripped rough microsomes from rat liver and outer membranes of nuclei from rat brain. These studies have revealed that membranes of the rough endoplasmic reticulum and nuclear envelope possess unique recognition and fusion properties and as such constitute the first demonstration of membrane interaction specificity at the intracellular level.

Proteomic analysis of the transitional endoplasmic reticulum in hepatocellular carcinoma: An organelle perspective on cancer

The transitional endoplasmic reticulum (tER) is composed of both rough and smooth ER membranes and thus participates in functions attributed to both these two subcellular compartments. In this paper we have compared the protein composition of tER isolated from dissected liver tumor nodules of aflatoxin B1-treated rats with that of tER from control liver. Tandem mass spectrometry (MS), peptide counts and immunoblot validation were used to identify and determine the relative expression level of proteins. Inhibitors of apoptosis (i.e. PGRMC1, tripeptidyl peptidase II), proteins involved in ribosome biogenesis (i.e. nucleophosmin, nucleolin), proteins involved in translation (i.e. eEF-2, and subunits of eIF-3), proteins involved in ubiquitin metabolism (i.e. proteasome subunits, USP10) and proteins involved in membrane traffic (i.e. SEC13-like 1, SEC23B, dynactin 1) were found overexpressed in tumor tER. Transcription factors (i.e. Pur-beta, BTF3) and molecular targets for C-Myc and NF-kappa B were observed overexpressed in tER from tumor nodules. Down-regulated proteins included cytochrome P450 proteins and enzymes involved in fatty acid metabolism and in steroid metabolism. Unexpectedly expression of the protein folding machinery (i.e. calreticulin) and proteins of the MHC class I peptide-loading complex did not change. Proteins of unknown function were detected in association with the tER and the novel proteins showing differential expression are potential new tumor markers. In many cases differential expression of proteins in tumor tER was comparable to that of corresponding genes reported in the Oncomine human database. Thus the molecular profile of tumor tER is different and this may confer survival advantage to tumor cells in cancer.

Terminal glycosylation in rat hepatic Golgi fractions: heterogeneous locations for sialic acid and galactose acceptors and their transferases.

Endogenous acceptors for N-acetylglucosamine (GlcNAc), galactose (Gal) or sialic acid (NeuAc) transfer were labeled to high activities when purified hepatic Golgi fractions were incubated with the corresponding radiolabeled nucleotide sugar in the absence of detergent. The in vitro conditions which were optimal for the endogenous glycosylation of GlcNAc and Gal acceptors (Mn2+, ATP) also promoted fusion within a subset of Golgi membranes. Electron microscope radioautography revealed that the majority of NeuAc acceptors were associated with unfused Golgi membranes, whereas the majority of Gal acceptors were localized to fused membranes. GlcNAc acceptors were approximately equally distributed between fused and unfused membranes. Under conditions in which Golgi membrane fusion was absent (-Mn2+), only NeuAc transfer was active. The majority of endogenous NeuAc acceptors were consequently assigned to the more trans regions of the hepatic Golgi apparatus as concluded from a combination of radioautography (NeuAc transfer) and acid NADPase cytochemistry (reactive medial and trans Golgi saccules). The distribution of NeuAc and Gal transferases was assessed after Percoll gradient centrifugation of disrupted Golgi fractions. The median density of NeuAc transferase was lower than that of Gal transferase. The studies are indicative of distinct Golgi components harboring the majority of acceptors and enzymes for terminal glycosylation.

Accumulation of polyunsaturated free fatty acids coincident with the fusion of rough endoplasmic reticulum membranes.

The accumulation of polyunsaturated free fatty acids (PUFAs) was observed coincident with GTP-dependent fusion of liver rough microsomes. Whereas 0.5 mM NADPH led to a parallel reduction (greater than 50%) in membrane fusion and PUFA accumulation, indomethacin (50 microM) either had little effect or slightly augmented both processes. CTP was observed to stimulate accumulation of PUFAs and diacylglycerol (DAG). Therefore PUFAs may be relevant for GTP-dependent membrane fusion and together with DAG may play a role in fusion stimulated in the presence of CTP.

Electrophoretic protein blots as aids in choosing fixatives for immunocytochemistry.

We used electrophoretic protein blots prepared from polyacrylamide gels to test the effect of different fixatives on the antigenicity of rough endoplasmic reticulum (RER) peptides from rat liver. Protein blots were prepared by the procedure of Towbin et al. (Proc Natl Acad Sci USA 76:4350, 1979), treated with different fixatives, rinsed to inactivate non-specific reactive sites, and then reacted with rabbit polyclonal anti-rat liver RER antibodies, followed by peroxidase-conjugated anti-rabbit antibodies. On the basis of differences in immunostaining densities as determined by densitometry, we found that RER peptides displayed differential sensitivities to various fixatives. Anti-rat liver RER antibodies and the immunogold technique were applied to methacrylate sections of in vitro fixed rat liver rough microsomes. Specific labeling was observed over the microsomes and was shown by quantitation to vary in a similar manner to the immunostaining of specific peptides in protein blots following different fixations. We conclude that protein blots may serve as useful tools for screening the effects of different fixatives on cell antigenicity, and therefore may be helpful in immunocytochemical studies.

Modulation of GTP-dependent fusion by linoleic and arachidonic acid in derivatives of rough endoplasmic reticulum from rat liver

The effect of modulation of the content of unsaturated free fatty acids on GTP-dependent fusion of stripped rough microsomes from rat liver was determined. Cytidine monophosphate, CDP and CTP were all observed to be able to stimulate free fatty acid accumulation and coincident membrane fusion. GTP was required for membrane fusion in the presence of cytidine nucleotide but was not required for free fatty acid accumulation. In the presence of GTP and cytidine nucleotide, the addition of ATP and CoA led to the synthesis of triacyglycerol and marked inhibition of both free fatty acid accumulation and membrane fusion. Delipidated bovine serum albumin also inhibited both free fatty acid accumulation and membrane fusion. Analysis by gas chromatography indicated that linoleic acid and arachidonic acid were the most actively fluctuating of the accumulated free fatty acids. Comparison by quantitation indicated a high correlation between GTP-dependent membrane fusion and changes in amount of unesterified linoleic acid and arachidonic acid. The results suggest that polyunsaturated free fatty acids may be required for GTP-dependent membrane fusion.