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␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors mediate the majority of excitatory signaling in the CNS, and the functional properties and subcellular fate of these receptors depend on receptor subunit composition. Subunit assembly is thought to occur in the endoplasmic reticulum (ER), although we are just beginning to understand the underlying mechanism. Here we examine the trafficking of Caenorhabditis elegans glutamate receptors through the ER. Our data indicate that neurons require signaling by the unfolded protein response (UPR) to move GLR-1, GLR-2, and GLR-5 subunits out of the ER and through the secretory pathway. In contrast, other neuronal transmembrane proteins do not require UPR signaling for ER exit. The requirement for the UPR pathway is cell type and age dependent: impairment for receptor trafficking increases as animals age and does not occur in all neurons. Expression of XBP-1, a component of the UPR pathway, is elevated in neurons during development. Our results suggest that UPR signaling is a critical step in neural function that is needed for glutamate receptor assembly and secretion. INTRODUCTIONIon channels conduct electrochemical signaling in the nervous system, and ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type ionotropic glutamate receptors (AMPAR) in particular mediate the bulk of excitatory transmission in the CNS. AMPAR subunits (up to 4 in mammals, referred to as GluR1-R4) are multi-transmembrane-spanning proteins that can assemble into tetrameric channels of differing subunit composition (Hollmann and Heinemann, 1994;Dingledine et al., 1999). The specific subunit composition of a given AMPAR channel plays a critical role in determining the functional properties of that channel, including its channel opening probability, ion selectivity, and cytosolic binding partners (Hollmann and Heinemann, 1994;Dingledine et al., 1999;Sheng, 2001;Shi et al., 2001;Malinow, 2003). Subunit composition can also control the subcellular localization and regulated cell biological fate of a channel (Beattie et al., 2000;Lin et al., 2000). For example, heteromeric complexes of GluR1-R2 receptors have been shown to be added to hippocampal synapses in an activity-dependent manner, whereas GluR2-R3 complexes appear to cycle into synaptic membranes in a constitutive manner (Passafaro et al., 2001;Shi et al., 2001). To better understand AMPAR function in the nervous system, it is important to determine how individual subunits assemble into specific complexes of channels.Multisubunit channel assembly in general is tied to the movement of channels through the secretory pathway. Most proteins exit the ER without the need for specialized export signals (Wieland et al., 1987). However, many channels and receptors seem to defy this general rule by requiring channel assembly, sometimes specific export signals, and sometimes the interaction of scaffolding proteins and certain signal transduction events before exiting the ER (Ma and Jan, 2002). One explanation is ...

The two major chemoreceptors of Escherichia coli, Tsr and Tar, mediate opposite responses to the same changes in cytoplasmic pH (pH i ). We set out to identify residues involved in pH i sensing to gain insight into the general mechanisms of signaling employed by the chemoreceptors. did not change the polarity but altered the time course of pH i response. These results suggest that the electrostatic properties of a short cytoplasmic region within the linker region that connects the second transmembrane helix to the first methylation helix is critical for switching the signaling state of the chemoreceptors during pH sensing. Similar conformational changes of this region in response to external ligands may be critical components of transmembrane signaling.Many biological processes, such as enzyme reactions and interactions between proteins, are influenced by pH. Therefore, cells have to sense and adapt to changes in extracellular and intracellular pH. Despite the accumulated knowledge about pH-dependent regulation in a wide variety of organisms, the molecular mechanisms of pH sensing are still poorly understood.Behavioral responses of Escherichia coli and Salmonella typhimurium to changes in pH provide a convenient system for studying the pH-sensing mechanism. These bacteria show repellent responses to weak acids and attractant responses to weak bases (1, 2). These responses are generated by decreases and increases of cytoplasmic pH (pH i ). 1 The changes in pH i were documented by 31 P nuclear magnetic resonance spectroscopy (3, 4). Usually, pH i in E. coli is maintained at around 7.5 over a range of extracellular pH (pH o ) values from 5.0 to 9.0 (3, 5). However, this strong pH i homeostasis can be disrupted by the addition of weak acids or weak bases to the culture medium. When pH o is lower than pH i , weak acids can traverse the membrane in their protonated (uncharged) form and release protons in the cytoplasm to decrease pH i . Similarly, when pH o is higher than pH i , weak bases can traverse the membrane in deprotonated (uncharged) forms and capture protons in the cytoplasm to increase pH i . These changes in pH i correlate well with tactic responses to weak acids and weak bases (4).The signal transduction pathway for chemotaxis in E. coli and S. typhimurium has been extensively studied at the molecular level (for reviews, see Refs. 6 -9). These organisms have a set of related methyl-accepting chemoreceptors that includes the serine receptor Tsr and the aspartate receptor Tar. These receptors have a remarkable ability to sense a variety of stimuli, including chemoattractants, chemorepellents, temperature, and pH.Tar and, presumably, the other chemoreceptors exist as a homodimer of about 60-kDa subunits (10). The dimeric cytoplasmic domains form stable complexes with the histidine kinase CheA and the adaptor protein CheW (11,12). Furthermore, the receptors, together with the CheA and CheW proteins, cluster at a cell pole (13).CheA phosphorylates itself and then serves as a phosphodonor for the response regul...

SummaryThe aspartate chemoreceptor (Tar) of Escherichia coli also serves as a thermosensor, and it is very amenable to genetic and biochemical analysis of the thermosensing mechanism. Its thermosensing properties are controlled by reversible methylation of the cytoplasmic signalling/adaptation domain of the protein. The unmethylated and the fully methylated (aspartatebound) receptors sense, as attractant stimuli, increases (warm sensor) and decreases (cold sensor) in temperature respectively. To learn more about the mechanism of thermosensing, we replaced the four methyl-accepting glutamyl residues with non-methylatable aspartyl residues in all possible combinations. In a strain defective in both methyltransferase (CheR) and methylesterase (CheB) activities, all of the mutant Tar proteins functioned as warm sensors. To create a situation in which all of the remaining glutamyl residues were methylated, we expressed the mutant proteins in a CheB-defective, CheR-overproducing strain. The fully glutamyl-methylated proteins were designed to mimic the full range of methylation states possible for wildtype Tar. Almost all of the methylated mutant receptors, including those with single glutamyl residues, were cold sensors in the presence of aspartate. Thus, binding of aspartate to Tar and methylation of its single glutamyl residue can invert its temperaturedependent signalling properties.

Calcium and calmodulin-dependent protein kinase II (CaMKII) plays a fundamental role in the synaptic plasticity events that underlie learning and memory. Regulation of CaMKII kinase activity occurs through an autoinhibitory mechanism in which a regulatory domain of the kinase occupies the catalytic site and calcium/calmodulin activates the kinase by binding to and displacing this regulatory domain. A single putative ortholog of CaMKII, encoded by unc-43, is present in the Caenorhabditis elegans nervous system. Here we examined UNC-43 subcellular localization in the neurons of intact animals and show that UNC-43 is localized to clusters in ventral cord neurites, as well as to an unlocalized pool within these neurites. A mutation that mimics autophosphorylation within the regulatory domain results in an increase in the levels of UNC-43 in the unlocalized neurite pool. Multiple residues of CaMKII facilitate the interaction between the catalytic domain and the regulatory domain, thereby keeping the kinase inactive. Whereas most mutations in these residues result in an increased neurite pool of UNC-43, we have identified two residues that result in the opposite effect when mutated: a decreased neurite pool of UNC-43. The activity of UNC-2, a voltage-dependent calcium channel, is also required for UNC-43 to accumulate in the neurites, suggesting that neural activity regulates the localization of UNC-43. Our results suggest that the activation of UNC-43 by calcium/calmodulin displaces the autoinhibitory domain, thereby exposing key residues of the catalytic domain that allow for protein translocation to the neurites.