Sara Francesca Colombo

Formation of stacked ER cisternae by low affinity protein interactions.

The endoplasmic reticulum (ER) can transform from a network of branching tubules into stacked membrane arrays (termed organized smooth ER [OSER]) in response to elevated levels of specific resident proteins, such as cytochrome b(5). Here, we have tagged OSER-inducing proteins with green fluorescent protein (GFP) to study OSER biogenesis and dynamics in living cells. Overexpression of these proteins induced formation of karmellae, whorls, and crystalloid OSER structures. Photobleaching experiments revealed that OSER-inducing proteins were highly mobile within OSER structures and could exchange between OSER structures and surrounding reticular ER. This indicated that binding interactions between proteins on apposing stacked membranes of OSER structures were not of high affinity. Addition of GFP, which undergoes low affinity, antiparallel dimerization, to the cytoplasmic domains of non–OSER-inducing resident ER proteins was sufficient to induce OSER structures when overexpressed, but addition of a nondimerizing GFP variant was not. These results point to a molecular mechanism for OSER biogenesis that involves weak homotypic interactions between cytoplasmic domains of proteins. This mechanism may underlie the formation of other stacked membrane structures within cells.

PI(4,5)P2-Dependent and Ca2+-Regulated ER-PM Interactions Mediated by the Extended Synaptotagmins

Most available information on endoplasmic reticulum (ER)-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca(2+) entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here, we show that the three extended synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca(2+) in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca(2+) regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca(2+) entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast) mediates the formation of ER-PM contacts sites, which are functionally distinct from those mediated by STIM1 and Orai1.

The tale of tail-anchored proteins: coming from the cytosol and looking for a membrane

A group of integral membrane proteins, known as C-tail anchored, is defined by the presence of a cytosolic NH2-terminal domain that is anchored to the phospholipid bilayer by a single segment of hydrophobic amino acids close to the COOH terminus. The mode of insertion into membranes of these proteins, many of which play key roles in fundamental intracellular processes, is obligatorily posttranslational, is highly specific, and may be subject to regulatory processes that modulate the proteins function. Although recent work has elucidated structural features in the tail region that determine selection of the correct target membrane, the molecular machinery involved in interpreting this information, and in modulating tail-anchored protein localization, has not been identified yet.

Transmembrane domain–dependent partitioning of membrane proteins within the endoplasmic reticulum

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein–protein and protein–lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail–anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER–Golgi boundary by simple receptor-independent mechanisms based on partitioning.

N-myristoylation determines dual targeting of mammalian NADH-cytochrome b(5) reductase to ER and mitochondrial outer membranes by a mechanism of kinetic partitioning

Mammalian NADH-cytochrome b(5) reductase (b5R) is an N-myristoylated protein that is dually targeted to ER and mitochondrial outer membranes. The N-linked myristate is not required for anchorage to membranes because a stretch of hydrophobic amino acids close to the NH2 terminus guarantees a tight interaction of the protein with the phospholipid bilayer. Instead, the fatty acid is required for targeting of b5R to mitochondria because a nonmyristoylated mutant is exclusively localized to the ER. Here, we have investigated the mechanism by which N-linked myristate affects b5R targeting. We find that myristoylation interferes with interaction of the nascent chain with signal recognition particle, so that a portion of the nascent chains escapes from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane. Thus, competition between two cotranslational events, binding of signal recognition particle and modification by N-myristoylation, determines the site of translation and the localization of b5R.

Biogenesis, trafficking and up-regulation of nicotinic ACh receptors.

Chronic nicotine exposure gives rise to neural adaptations that change whole cell physiology and behaviour mainly by interacting with neuronal nicotinic acetylcholine receptors (nAChRs). The major nicotine-induced neuroadaptation is the up-regulation of brain nAChRs by means of cell-delimited post-translational mechanisms. We review what is known of the processes regulating nAChR assembly, degradation and trafficking, and how nicotine-induced modulation of these processes leads to nAChR up-regulation and changes in downstream neuronal plasticity at molecular, cellular and circuit level.

The role of cytosolic proteins in the insertion of tail-anchored proteins into phospholipid bilayers.

Tail-anchored (TA) proteins are membrane proteins that contain an N-terminal domain exposed to the cytosol and a single transmembrane segment near the C-terminus followed by few or no polar residues. TA proteins with a mildly hydrophobic transmembrane domain, such as cytochrome b5 (b5), are able to insert post-translationally into pure lipid vesicles without assistance from membrane proteins. Here, we investigated whether any cytosolic proteins are needed to maintain b5 in a competent state for transmembrane integration. Using b5 constructs translated in vitro or produced in bacteria, we demonstrate that cytosolic proteins are neither necessary nor facilitatory for the unassisted translocation of b5. Furthermore, we demonstrate that no cytosolic protein is involved in the translocation of a C-terminal domain of 85 residues appended to the transmembrane domain of b5. Nevertheless, b5 does bind cytosolic proteins, and in their presence but not in their absence, its insertion into liposomes is inhibited by the thiol oxidant diamide and the alkylating agent N-ethylmaleimide. The effect of diamide is also observed in living cells. Thus, the specific in vivo targeting of b5 might be achieved by interaction with redox-sensitive targeting factors that hinder its nonspecific insertion into any permissive bilayer.

Transcription Factor PHOX2A Regulates the Human α3 Nicotinic Receptor Subunit Gene Promoter

PHOX2A is a paired-like homeodomain transcription factor that participates in specifying the autonomic nervous system. It is also involved in the transcriptional control of the noradrenergic neurotransmitter phenotype as it regulates the gene expression of tyrosine hydroxylase and dopamine-β-hydroxylase. The results of this study show that the human orthologue of PHOX2A is also capable of regulating the transcription of the human α3 nicotinic acetylcholine receptor gene, which encodes the ligand-binding subunit of the ganglionic type nicotinic receptor. In particular, we demonstrated by chromatin immunoprecipitation and DNA pulldown assays that PHOX2A assembles on the SacI-NcoI region of α3 promoter and, by co-transfection experiments, that it exerts its transcriptional effects by acting through the 60-bp minimal promoter. PHOX2A does not seem to bind to DNA directly, and its DNA binding domain seems to be partially dispensable for the regulation of α3 gene transcription. However, as suggested by the findings of our co-immunoprecipitation assays, it may establish direct or indirect protein-protein interactions with Sp1, thus regulating the expression of α3 through a DNA-independent mechanism. As the α3 subunit is expressed in every terminally differentiated ganglionic cell, this is the first example of a “pan-autonomic” gene whose expression is regulated by PHOX2 proteins.

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. Here, using transfected mammalian cells as model system, we characterized the response of the human α3β4 receptor subtype to nicotine and the mechanism of action of the drug. Nicotine, when present at 1 mm concentration, elicited a ∼5-fold increase of cell surface α3β4 and showed a more modest upregulatory effect also at concentrations as low as 10 μM. Upregulation was obtained if nicotine was present during, but not after, pentamer assembly and was caused by increased stability and trafficking of receptors assembled in the presence of the drug. Experimental determinations as well as computational studies of subunit stoichiometry showed that nicotine favors assembly of pentamers with (α3)2(β4)3 stoichiometry; these are less prone than (α3)3(β4)2 receptors to proteasomal degradation and, because of the presence in the β subunit of an endoplasmic reticulum export motif, more efficiently transported to the plasma membrane. Our findings uncover a novel mechanism of nicotine-induced α3β4 nAChR upregulation that may be relevant also for other nAChR subtypes.

CDK5 Regulatory Subunit-associated Protein 1-Like 1 (CDKAL1) Is a Tail-anchored Protein in the Endoplasmic Reticulum (ER) of Insulinoma Cells

Background: Cdkal1 is a type 2 diabetes (T2D) susceptibility gene responsible for tRNALys modification. Results: CDKAL1 is a tail-anchored protein inserted in the ER via the TRC40/Get3 pathway. Its down-regulation affects the expression of some insulin granule proteins and the ER stress marker CHOP10. Conclusion: CDKAL1 participates in translation of insulin granule proteins and ER stress. Significance: Characterizing CDKAL1 contributes to T2D research. Genome-wide association studies have led to the identification of numerous susceptibility genes for type 2 diabetes. Among them is Cdkal1, which is associated with reduced β-cell function and insulin release. Recently, CDKAL1 has been shown to be a methylthiotransferase that modifies tRNALys to enhance translational fidelity of transcripts, including the one encoding proinsulin. Here, we report that out of several CDKAL1 isoforms deposited in public databases, only isoform 1, which migrates as a 61-kDa protein by SDS-PAGE, is expressed in human islets and pancreatic insulinoma INS-1 and MIN6 cells. We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. Using endo-β-N-acetylglucosaminidase H and peptide:N-glycosidase F sensitivity assays on CDKAL1 constructs carrying an N-glycosylation site within the luminal domain, we further established that CDKAL1 is an endoplasmic reticulum-resident protein. Moreover, we observed that silencing CDKAL1 in INS-1 cells reduces the expression of secretory granule proteins prochromogranin A and proICA512/ICA512-TMF, in addition to proinsulin and insulin. This correlated with reduced glucose-stimulated insulin secretion. Taken together, our findings provide new insight into the role of CDKAL1 in insulin-producing cells and help to understand its involvement in the pathogenesis of diabetes.