A role for mitochondria-ER crosstalk in amyotrophic lateral sclerosis 8 pathogenesis.

Protein aggregates in motoneurons, a pathological hallmark of amyotrophic lateral sclerosis, have been suggested to play a key pathogenetic role. ALS8, characterized by ER-associated inclusions, is caused by a heterozygous mutation in VAPB, which acts at multiple membrane contact sites between the ER and almost all other organelles. The link between protein aggregation and cellular dysfunction is unclear.

Chemical chaperones in metabolic fitness beyond protein folding.

Chemical chaperones are small molecules that improve protein folding, alleviating aberrant pathological phenotypes due to protein misfolding. Recent reports suggest that, in parallel with their role in relieving endoplasmic reticulum (ER) stress, chemical chaperones rescue mitochondrial function and insulin signaling. These effects may underlie their pharmacological action on metabolically demanding tissues.

ERO1 alpha deficiency impairs angiogenesis by increasing N-glycosylation of a proangiogenic VEGFA.

N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each other. Here, to analyze crosstalk between N-glycosylation and oxidation, we investigated how the protein disulfide oxidase ERO1-alpha affects glycosylation of the angiogenic VEGF, a key regulator of vascular homeostasis. ERO1 deficiency, while retarding disulfide bond formation in VEGF, increased utilization of its single N-glycosylation sequon, which lies close to an intra-polypeptide disulfide bridge, and concomitantly slowed its secretion.

The Link between VAPB Loss of Function and Amyotrophic Lateral Sclerosis.

The VAP proteins are integral adaptor proteins of the endoplasmic reticulum (ER) membrane that recruit a myriad of interacting partners to the ER surface. Through these interactions, the VAPs mediate a large number of processes, notably the generation of membrane contact sites between the ER and essentially all other cellular membranes. In 2004, it was discovered that a mutation (p.

Mutant VAPB: Culprit or Innocent Bystander of Amyotrophic Lateral Sclerosis?

Nearly twenty years ago a mutation in the VAPB gene, resulting in a proline to serine substitution (p.P56S), was identified as the cause of a rare, slowly progressing, familial form of the motor neuron degenerative disease Amyotrophic Lateral Sclerosis (ALS). Since then, progress in unravelling the mechanistic basis of this mutation has proceeded in parallel with research on the VAP proteins and on their role in establishing membrane contact sites between the ER and other organelles.

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity.

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain.

Searching for remote homologs of CAML among eukaryotes.

The tryptophan rich basic protein/calcium signal-modulating cyclophilin ligand (WRB/CAML) and Get1p/Get2p complexes, in vertebrates and yeast, respectively, mediate the final step of tail-anchored protein insertion into the endoplasmic reticulum membrane via the Get pathway. While WRB appears to exist in all eukaryotes, CAML homologs were previously recognized only among chordates, raising the question as to how CAML's function is performed in other phyla. Furthermore, whereas WRB was recognized as the metazoan homolog of Get1, CAML and Get2, although functionally equivalent, were not considered to be homologous.

The WRB Subunit of the Get3 Receptor is Required for the Correct Integration of its Partner CAML into the ER.

Calcium-modulating cyclophilin ligand (CAML), together with Tryptophan rich basic protein (WRB, Get1 in yeast), constitutes the mammalian receptor for the Transmembrane Recognition Complex subunit of 40 kDa (TRC40, Get3 in yeast), a cytosolic ATPase with a central role in the post-translational targeting pathway of tail-anchored (TA) proteins to the endoplasmic reticulum (ER) membrane. CAML has also been implicated in other cell-specific processes, notably in immune cell survival, and has been found in molar excess over WRB in different cell types. Notwithstanding the stoichiometric imbalance, WRB and CAML depend strictly on each other for expression.

The Ways of Tails: the GET Pathway and more.

Due to their topology tail-anchored (TA) proteins must target to the membrane independently of the co-translational route defined by the signal sequence recognition particle (SRP), its receptor and the translocon Sec61. More than a decade of work has extensively characterized a highly conserved pathway, the yeast GET or mammalian TRC40 pathway, which is capable of countering the biogenetic challenge posed by the C-terminal TA anchor. In this review we briefly summarize current models of this targeting route and focus on emerging aspects such as the intricate interplay with the proteostatic network of cells and with other targeting pathways.

VAPB depletion alters neuritogenesis and phosphoinositide balance in motoneuron-like cells: relevance to VAPB-linked amyotrophic lateral sclerosis.

VAPB and VAPA are ubiquitously expressed endoplasmic reticulum membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of amyotrophic lateral sclerosis; however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted.

The GET pathway can increase the risk of mitochondrial outer membrane proteins to be mistargeted to the ER.

Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It has been widely observed that some mitochondrial outer membrane (MOM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked.

The fifth subunit in α3β4 nicotinic receptor is more than an accessory subunit.

The α3β4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 β4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the β4 and α3 subunit sequences, with different monomeric subunits.

Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail-anchored proteins.

Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively.

CAML mediates survival of Myc-induced lymphoma cells independent of tail-anchored protein insertion.

Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions, along with WRB and TRC40, to mediate tail-anchored (TA) protein insertion into the ER membrane. Physiologic roles for CAML include endocytic trafficking, intracellular calcium signaling, and the survival and proliferation of specialized immune cells, recently attributed to its requirement for TA protein insertion. To identify a possible role for CAML in cancer cells, we generated transgenic mice that carry a tamoxifen-inducible deletion allele of .

Tail-anchored Protein Insertion in Mammals: FUNCTION AND RECIPROCAL INTERACTIONS OF THE TWO SUBUNITS OF THE TRC40 RECEPTOR.

The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells.

Getting membrane proteins on and off the shuttle bus between the endoplasmic reticulum and the Golgi complex.

Secretory proteins exit the endoplasmic reticulum (ER) in coat protein complex II (COPII)-coated vesicles and then progress through the Golgi complex before delivery to their final destination. Soluble cargo can be recruited to ER exit sites by signal-mediated processes (cargo capture) or by bulk flow. For membrane proteins, a third mechanism, based on the interaction of their transmembrane domain (TMD) with lipid microdomains, must also be considered.

Autophagy and Neurodegeneration: Insights from a Cultured Cell Model of ALS.

Autophagy plays a major role in the elimination of cellular waste components, the renewal of intracellular proteins and the prevention of the build-up of redundant or defective material. It is fundamental for the maintenance of homeostasis and especially important in post-mitotic neuronal cells, which, without competent autophagy, accumulate protein aggregates and degenerate. Many neurodegenerative diseases are associated with defective autophagy; however, whether altered protein turnover or accumulation of misfolded, aggregate-prone proteins is the primary insult in neurodegeneration has long been a matter of debate.

Amyotrophic lateral sclerosis-linked mutant VAPB inclusions do not interfere with protein degradation pathways or intracellular transport in a cultured cell model.

VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome.

A positive signal prevents secretory membrane cargo from recycling between the Golgi and the ER.

The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward-bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells.

Visualization of endoplasmic reticulum subdomains in cultured cells.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions.

Nicotine-modulated subunit stoichiometry affects stability and trafficking of α3β4 nicotinic receptor.

Heteromeric nAChRs are pentameric cation channels, composed of combinations of two or three α and three or two β subunits, which play key physiological roles in the central and peripheral nervous systems. The prototypical agonist nicotine acts intracellularly to upregulate many nAChR subtypes, a phenomenon that is thought to contribute to the nicotine dependence of cigarette smokers. The α3β4 subtype has recently been genetically linked to nicotine dependence and lung cancer; however, the mode of action of nicotine on this receptor subtype has been incompletely investigated.