The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

The exit of low‐density lipoprotein derived cholesterol (LDL‐C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann–Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol‐binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N‐terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well‐established role in disassembling the ESCRT (endosomal sorting complex required for transport)‐III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL‐C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT‐III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT‐III.     

Trafficking of the IKs‐Complex in MDCK Cells: Site of Subunit Assembly and Determinants of Polarized Localization

The voltage‐gated potassium channel K_V7.1 is regulated by non‐pore forming regulatory KCNE β‐subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current I_Ks. However, where the subunits assemble and which of the subunits determines localization of the I_Ks‐complex has not been unequivocally resolved yet. We employed trafficking‐deficient K_V7.1 and KCNE1 mutants to investigate I_Ks trafficking using the polarized Madin‐Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes place in a post‐endoplasmic reticulum compartment. We demonstrate that K_V7.1 targets the I_Ks‐complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical K_V7.1 basolateral targeting signals. Our data provide a possible explanation to the fact that K_V7.1 can be localized apically or basolaterally in different epithelial tissues and offer a solution to divergent literature results regarding the effect of KCNE subunits on the subcellular localization of K_V7.1/KCNE complexes .     

Lysosomal Membrane Proteins and Their Central Role in Physiology

The lysosomal membrane was thought for a long time to primarily act as a physical barrier separating the luminal acidic milieu from the cytoplasmic environment. Meanwhile, it has been realized that unique lysosomal membranes play essential roles in a number of cellular events ranging from phagocytosis, autophagy, cell death, virus infection to membrane repair. This review provides an overview about the most interesting emerging functions of lysosomal membrane proteins and how they contribute to health and disease. Their importance is exemplified by their role in acidification, transport of metabolites and ions across the membrane, intracellular transport of hydrolases and the regulation of membrane fusion events. Studies in patient cells, non‐mammalian model organisms and knockout mice contributed to our understanding of how the different lysosomal membrane proteins affect cellular homeostasis, developmental processes as well as tissue functions. Because these proteins are central for the biogenesis of this compartment they are also considered as attractive targets to modulate the lysosomal machinery in cases where impaired lysosomal degradation leads to cellular pathologies. We are only beginning to understand the complex composition and function of these proteins which are tightly linked to processes occurring throughout the endocytic and biosynthetic pathways.      

Toward an integrative view of Optineurin functions

This review highlights recent advances in our understanding of the mechanisms of Optineurin (Optn) action and its implication in diseases. Optn has emerged as a key player regulating various physiological processes, including membrane trafficking, protein secretion, cell division and host defense against pathogens. Furthermore, there is growing evidence for an association of Optn mutations with human diseases such as primary open-angle glaucoma, amyotrophic lateral sclerosis and Paget’s disease of bone. Optn functions depend on its precise subcellular localization and its interaction with other proteins. Here, we review the mechanisms that allow Optn to ensure a timely and spatially coordinated integration of different physiological processes and discuss how their deregulation may lead to different pathologies.     

Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

Abstract

With each heartbeat, billions of cardiomyocytes work in concert to propagate the electrical excitation needed to effectively circulate blood. Regulated expression and timely delivery of connexin proteins to form gap junctions at the specialized cell–cell contact region, known as the intercalated disc, is essential to ventricular cardiomyocyte coupling. We focus this review on several regulatory mechanisms that have been recently found to govern the lifecycle of connexin 43 (Cx43), the short-lived and most abundantly expressed connexin in cardiac ventricular muscle. The Cx43 lifecycle begins with gene expression, followed by oligomerization into hexameric channels, and then cytoskeletal-based transport toward the disc region. Once delivered, hemichannels interact with resident disc proteins and are organized to effect intercellular coupling. We highlight recent studies exploring regulation of Cx43 localization to the intercalated disc, with emphasis on alternatively translated Cx43 isoforms and cytoskeletal transport machinery that together regulate Cx43 gap junction coupling between cardiomyocytes.     

Gap Junction Assembly: Multiple Connexin Fluorophores Identify Complex Trafficking Pathways

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.     

Ubiquilin-1 and protein quality control in Alzheimer disease

Single nucleotide polymorphisms in the ubiquilin-1 gene may confer risk for late-onset Alzheimer disease (AD). We have shown previously that ubiquilin-1 functions as a molecular chaperone for the amyloid precursor protein (APP) and that protein levels of ubiquilin-1 are decreased in the brains of AD patients. We have recently found that ubiquilin-1 regulates APP trafficking and subsequent secretase processing by stimulating non-degradative ubiquitination of a single lysine residue in the cytosolic domain of APP. Thus, ubiquilin-1 plays a central role in regulating APP biosynthesis, trafficking and ultimately toxicity. As ubiquilin-1 and other ubiquilin family members have now been implicated in the pathogenesis of numerous neurodegenerative diseases, these findings provide mechanistic insights into the central role of ubiquilin proteins in maintaining neuronal proteostasis.     

Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction

The Akt family of serine‐threonine kinases integrates a myriad of signals governing cell proliferation, apoptosis, glucose metabolism, and cytoskeletal organization. Akt affects neuronal morphology and function, influencing dendrite growth and the expression of ion channels. Akt is also an integral element of PI3Kinase‐target of rapamycin (TOR)‐Rheb signaling, a pathway that affects synapse assembly in both vertebrates and Drosophila. Our recent findings demonstrated that disruption of this pathway in Drosophila is responsible for a number of neurodevelopmental deficits that may also affect phenotypes associated with tuberous sclerosis complex, a disorder resulting from mutations compromising the TSC1/TSC2 complex, an inhibitor of TOR (Dimitroff et al., 2012). Therefore, we examined the role of Akt in the assembly and physiological function of the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse that displays developmental and activity‐dependent plasticity. The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR). Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin, a Drosophila t‐SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA. Our findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration. © 2013 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 73: 723–743, 2013