visualization-of-endoplasmic-reticulum-subdomains-in-cultured-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 ...

University of Milan

National Research Council (CNR)

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 tRNA(Lys) 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.

CDK5 regulatory subunit-associated protein 1-like 1 (CDKAL1) is a tail-anchored protein in the endoplasmic reticulum (ER) of insulinoma cells.

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.

PI(4,5)P(2)-dependent and Ca(2+)-regulated ER-PM interactions mediated by the extended synaptotagmins.

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.

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

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.

Article	Year
CDK5 regulatory subunit-associated protein 1-like 1 (CDKAL1) is a tail-anchored protein in the endoplasmic reticulum (ER) of insulinoma cells. The Journal of biological chemistry\| PubMed ID: 23048041	2012
PI(4,5)P(2)-dependent and Ca(2+)-regulated ER-PM interactions mediated by the extended synaptotagmins. Cell\| PubMed ID: 23791178	2013
Biogenesis, trafficking and up-regulation of nicotinic ACh receptors. Biochemical pharmacology\| PubMed ID: 23830821	2013
Nicotine-modulated subunit stoichiometry affects stability and trafficking of α3β4 nicotinic receptor. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 23884938	2013

Sara Francesca Colombo

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