The N-terminal region of Jaw1 has a role to inhibit the formation of organized smooth endoplasmic reticulum as an intrinsically disordered region.

Jaw1/LRMP is a type II integral membrane protein that is localized at the endoplasmic reticulum (ER) and outer nuclear membrane. We previously reported that a function of Jaw1 is to maintain the nuclear shape as a KASH protein via its carboxyl terminal region, a component of linker of nucleoskeleton and cytoskeleton complex in the oligomeric state. Although the oligomerization of some KASH proteins via the cytosolic regions serves to stabilize protein-protein interactions, the issue of how the oligomerization of Jaw1 is regulated is not completely understood. Therefore, we focused on three distinct regions on the cytosolic face of Jaw1: the N-terminal region, the coiled-coil domain and the stem region, in terms of oligomerization. A co-immunoprecipitation assay showed that its coiled-coil domain is a candidate for the oligomerization site. Furthermore, our data indicated that the N-terminal region prevents the aberrant oligomerization of Jaw1 as an intrinsically disordered region (IDR). Importantly, the ectopic expression of an N-terminal region deleted mutant caused the formation of organized smooth ER (OSER), structures such as nuclear karmellae and whorls, in B16F10 cells. Furthermore, this OSER interfered with the localization of the oligomer and interactors such as the type III inositol 1,4,5-triphosphate receptor (IP3R3) and SUN2. In summary, the N-terminal region of Jaw1 inhibits the formation of OSER as an IDR to maintain the homeostatic localization of interactors on the ER membrane.

OATP1B3-1B7 (LST-3TM12) Is a Drug Transporter That Affects Endoplasmic Reticulum Access and the Metabolism of Ezetimibe.

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe ß-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.

Quantitative proteomic survey of endoplasmic reticulum in mouse liver.

To gain a better understanding of the critical function of the endoplasmic reticulum (ER) in liver, we carried out a proteomic survey of mouse liver ER. The ER proteome was profiled with a new three-dimensional, gel-based strategy. From 6152 and 6935 MS spectra, 903 and 1042 proteins were identified with at least two peptides matches at 95% confidence in the rough (r) and smooth (s) ER, respectively. Comparison of the rER and sER proteomes showed that calcium-binding proteins are significantly enriched in the sER suggesting that the ion-binding function of the ER is compartmentalized. Comparison of the rat and mouse ER proteomes showed that 662 proteins were common to both, comprising 53.5% and 49.3% of those proteomes, respectively. We proposed that these proteins were stably expressed proteins that were essential for the maintenance of ER function. GO annotation with a hypergeometric model proved this hypothesis. Unexpectedly, 210 unknown proteins and some proteins previously reported to occur in the cytosol were highly enriched in the ER. This study provides a reference map for the ER proteome of liver. Identification of new ER proteins will enhance our current understanding of the ER and also suggest new functions for this organelle.

Infectious bronchitis virus 3a protein localizes to a novel domain of the smooth endoplasmic reticulum.

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.

Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae.

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

Calcium regulates the association between mitochondria and a smooth subdomain of the endoplasmic reticulum.

Association between the ER and mitochondria has long been observed, and the formation of close contacts between ER and mitochondria is necessary for the ER-mediated sequestration of cytosolic calcium by mitochondria. Autocrine motility factor receptor (AMF-R) is a marker for a smooth subdomain of the ER, shown here by confocal microscopy to be distinct from, yet closely associated with the calnexin- or calreticulin-labeled ER. By EM, smooth ER AMF-R tubules exhibit direct interactions with mitochondria, identifying them as a mitochondria-associated smooth ER subdomain. In digitonin-permeabilized MDCK cells, the addition of rat liver cytosol stimulates the dissociation of smooth ER and mitochondria under conditions of low calcium. Using BAPTA chelators of various affinities and CaEGTA buffers of defined free Ca(2+) concentrations and quantitative confocal microscopy, we show that free calcium concentrations <100 nM favor dissociation, whereas those >1 microM favor close association between these two organelles. Therefore, we describe a cellular mechanism that facilitates the close association of this smooth ER subdomain and mitochondria when cytosolic free calcium rises above physiological levels.

Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth endoplasmic reticulum membrane dynamics.

The endoplasmic reticulum (ER) consists of subcompartments that have distinct protein constituents, morphological appearances, and functions. To understand the mechanisms that regulate the intricate and dynamic organization of the endoplasmic reticulum, it is important to identify and characterize the molecular machinery involved in the assembly and maintenance of the different subcompartments. Here we report that syntaxin 17 is abundantly expressed in steroidogenic cell types and specifically localizes to smooth membranes of the ER. By immunoprecipitation analyses, syntaxin 17 exists in complexes with a syntaxin regulatory protein, rsly1, and/or two intermediate compartment SNARE proteins, rsec22b and rbet1. Furthermore, we found that syntaxin 17 is anchored to the smooth endoplasmic reticulum through an unusual mechanism, requiring two adjacent hydrophobic domains near its carboxyl terminus. Converging lines of evidence indicate that syntaxin 17 functions in a vesicle-trafficking step to the smooth-surfaced tubular ER membranes that are abundant in steroidogenic cells.

Selective localization of Bcl-2 to the inner mitochondrial and smooth endoplasmic reticulum membranes in mammalian cells.

Bcl-2, an anti-apoptotic protein, is believed to be localized in the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope. However, Bcl-2 has also been suggested as playing a role in the maintenance of mitochondrial membrane potential, indicating its possible association with the inner mitochondrial membrane. We therefore further examined the exact localization of Bcl-2 in mitochondria purified from wild-type and bcl-2-transfected PC12 cells and pre- and postnatal rat brains. Double immunostaining demonstrated that Bcl-2 was co-localized with subunit beta of F1F0ATPase in the inner mitochondrial membrane. Biochemical analysis of isolated mitochondria using digitonin and trypsin suggests an association of Bcl-2 with the inner mitochondrial membrane. More interestingly, the majority of Bcl-2 disappeared from the inner membrane of mitochondria when cultured under serum deprivation. These results suggest that Bcl-2 acts as an anti-apoptotic regulator by localizing mainly to the inner mitochondrial and smooth ER membranes.

Mitsugumin23, a novel transmembrane protein on endoplasmic reticulum and nuclear membranes.

We report the identification using monoclonal antibody and the primary structure by cDNA cloning of mitsugumin23, a novel transmembrane protein with a molecular mass of approximately 23 kDa from skeletal muscle sarcoplasmic reticulum. Mitsugumin23 possesses three putative transmembrane segments, and its carboxy-terminal hydrophilic region exhibits sequence similarity with the tail-end portion of the myosin heavy chain. Immunochemical analysis showed that this protein is distributed throughout the outer nuclear membrane and the sarcoplasmic reticulum including the terminal cisternae at the triad junction in skeletal muscle cells. Furthermore, RNA blotting and immunohistochemical experiments demonstrated that mitsugumin23 is distributed among a wide variety of cell types in various tissues. The distribution and primary structure indicate the possibility that mitsugumin23 interacts with cytoplasmic protein(s) and participates in a housekeeping function on the intracellular organelle membranes.

Ultrastructural localization of sorcin, a 22 kDa calcium binding protein, in the rat caudate-putamen nucleus: association with ryanodine receptors and intracellular calcium release.

Sorcin is a 22 kDa calcium binding protein that is widely distributed in mammalian tissues, including brain, and is associated with the ryanodine receptor (RyR) family of intracellular calcium-release channels in the heart. To determine the cellular sites for potential central functions of sorcin, we examined the electron microscopic immunocytochemical localization of antipeptide antisera against sorcin and against cardiac and brain RyR in the rat caudate-putamen nucleus (CPN), one of the few regions expressing high levels of brain RyR. Sorcin-like immunoreactivity (S-LI) was detected in both neurons and glia by using immunoperoxidase and immunogold methods. Of 1,735 profiles containing immunogold-silver labeling for sorcin, almost 50% were dendrites and many of these dendrites were spiny. The remainder were mainly small axons, axon terminals, and, more rarely, glia. Furthermore, analysis of dually labeled tissue sections showed the presence of sorcin in many of the dendrites and some of the axonal and glial processes containing RyR. In dendrites, gold-silver deposits showing S-LI were prominently localized to saccules of smooth endoplasmic reticulum and mitochondria, both of which are known to store calcium. These labeled structures were located near the plasma membrane at sites postsynaptic to excitatory-type asymmetric junctions, as well as non-synaptic portions of the plasma membrane. In axons, S-LI was also often seen at extrasynaptic sites on, or near, the plasma membrane. We conclude that in the rat CPN, sorcin may act independently or, in conjunction with RyR, to modulate cytoplasmic release of calcium, mainly from smooth endoplasmic reticulum and/or mitochondria in neurons.

The organization of the endoplasmic reticulum and the intermediate compartment in cultured rat hippocampal neurons.

The boundaries of the organelles of the biosynthetic endomembrane system are still controversial. In this paper we take advantage of the unique architectural organization of neurons to investigate the localization of a spectrum of compartment-specific markers with the goal of defining the location of the rough endoplasmic reticulum (ER), smooth ER, intermediate compartment, and the Golgi complex. Markers of the rough ER (signal sequence receptor), Golgi complex (mannosidase II), and the trans Golgi network (TGN38) were essentially restricted to the cell body and the initial segment of one of the cell's dendrites. In contrast the cytochemical reaction product for glucose 6 phosphate, a classical ER marker, in addition to staining ER structures in the cell body also reacted with smooth ER elements that extended into both axons and dendrites. These peripheral smooth ER elements also reacted at the immunofluorescence level for ER marker 3-hydroxy-3-methylglutaryl-coenzyme A reductase, as well as for calnexin and protein disulfide isomerase. We also analyzed the location of rab1, rab2, p58, the KDEL receptor, and beta-subunit of coatomer. These intermediate compartment markers were found predominantly in the cell body but also extended to the proximal parts of the dendrites. Collectively, our data argue that the ER of hippocampal neurons consists of functionally and spatially distinct and separated domains, and they stress the power of the hippocampal neuron system for investigations of the organization of the ER by light microscopy.