dPob/EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors.

In eukaryotes, most integral membrane proteins are synthesized, integrated into the membrane, and folded properly in the endoplasmic reticulum (ER). We screened the mutants affecting rhabdomeric expression of rhodopsin 1 (Rh1) in the Drosophila photoreceptors and found that dPob/EMC3, EMC1, and EMC8/9, Drosophila homologs of subunits of ER membrane protein complex (EMC), are essential for stabilization of immature Rh1 in an earlier step than that at which another Rh1-specific chaperone (NinaA) acts. dPob/EMC3 localizes to the ER and associates with EMC1 and calnexin. Moreover, EMC is required for the stable expression of other multi-pass transmembrane proteins such as minor rhodopsins Rh3 and Rh4, transient receptor potential, and Na(+)K(+)-ATPase, but not for a secreted protein or type I single-pass transmembrane proteins. Furthermore, we found that dPob/EMC3 deficiency induces rhabdomere degeneration in a light-independent manner. These results collectively indicate that EMC is a key factor in the biogenesis of multi-pass transmembrane proteins, including Rh1, and its loss causes retinal degeneration.

GPI biosynthesis is essential for rhodopsin sorting at the trans-Golgi network in Drosophila photoreceptors.

Sorting of integral membrane proteins plays crucial roles in establishing and maintaining the polarized structures of epithelial cells and neurons. However, little is known about the sorting mechanisms of newly synthesized membrane proteins at the trans-Golgi network (TGN). To identify which genes are essential for these sorting mechanisms, we screened mutants in which the transport of Rhodopsin 1 (Rh1), an apical integral membrane protein in Drosophila photoreceptors, was affected. We found that deficiencies in glycosylphosphatidylinositol (GPI) synthesis and attachment processes cause loss of the apical transport of Rh1 from the TGN and mis-sorting to the endolysosomal system. Moreover, Na(+)K(+)-ATPase, a basolateral membrane protein, and Crumbs (Crb), a stalk membrane protein, were mistransported to the apical rhabdomeric microvilli in GPI-deficient photoreceptors. These results indicate that polarized sorting of integral membrane proteins at the TGN requires the synthesis and anchoring of GPI-anchored proteins. Little is known about the cellular biological consequences of GPI deficiency in animals in vivo. Our results provide new insights into the importance of GPI synthesis and aid the understanding of pathologies involving GPI deficiency.

Brain-derived neurotrophic factor-mediated retrograde signaling required for the induction of long-term potentiation at inhibitory synapses of visual cortical pyramidal neurons.

High-frequency stimulation (HFS) induces long-term potentiation (LTP) at inhibitory synapses of layer 5 pyramidal neurons in developing rat visual cortex. This LTP requires postsynaptic Ca2+ rise for induction, while the maintenance mechanism is present at the presynaptic site, suggesting presynaptic LTP expression and the necessity of retrograde signaling. We investigated whether the supposed signal is mediated by brain-derived neurotrophic factor (BDNF), which is expressed in pyramidal neurons but not inhibitory interneurons. LTP did not occur when HFS was applied in the presence of the Trk receptor tyrosine kinase inhibitor K252a in the perfusion medium. HFS produced LTP when bath application of K252a was started after HFS or when K252a was loaded into postsynaptic cells. LTP did not occur in the presence of TrkB-IgG scavenging BDNF or function-blocking anti-BDNF antibody in the medium. In cells loaded with the Ca2+ chelator BAPTA, the addition of BDNF to the medium enabled HFS to induce LTP without affecting baseline synaptic transmission. These results suggest that BDNF released from postsynaptic cells activates presynaptic TrkB, leading to LTP. Because BDNF, expressed activity dependently, regulates the maturation of cortical inhibition, inhibitory LTP may contribute to this developmental process, and hence experience-dependent functional maturation of visual cortex.

High-frequency stimulation together with adrenoceptor activation facilitates the maintenance of long-term potentiation at visual cortical inhibitory synapses.

Long-term potentiation (LTP) at inhibitory synapses of rat visual cortex requires firing of presynaptic cells for maintenance, at least at a low frequency. We examined the roles of adrenoceptors in this LTP maintenance. Although high-frequency stimulation (HFS) failed to produce LTP in normal Ca2+ medium, it produced pathway-specific LTP with addition of noradrenaline to the medium soon after HFS. However, this LTP disappeared after washout of noradrenaline. HFS applied during noradrenaline application produced LTP persisting even after washout, indicating that HFS together with adrenoceptor activation makes the adrenergic facilitation enduring. After washout, LTP was produced further by HFS of the conditioned, but not the unconditioned, pathway by the first HFS. Pharmacological examination demonstrated that alpha2 and beta, but not alpha1, receptors facilitated LTP maintenance synergistically. Bath application, but not postsynaptic loading, of either the adenylyl cyclase activator forskolin or the protein kinase C (PKC) activator phorbol ester facilitated LTP maintenance. These results suggest that adrenergic facilitation of LTP maintenance is mediated by presynaptic adrenoceptors via a subfamily of adenylyl cyclases stimulated by Gsalpha, Gibetagamma, and PKC. Thus, it is likely that the activity of noradrenergic afferents takes part in the control of LTP duration at visual cortical inhibitory synapses.