A conserved viral amphipathic helix governs the replication site-specific membrane association

Positive-strand RNA viruses assemble their viral replication complexes (VRCs) on specific host organelle membranes, yet it is unclear how viral replication proteins recognize and what motifs or domains in viral replication proteins determine their localizations. We show here that an amphipathic helix, helix B in replication protein 1a of brome mosaic virus (BMV), is necessary for 1a's localization to the nuclear endoplasmic reticulum (ER) membrane where BMV assembles its VRCs. Helix B is also sufficient to target soluble proteins to the nuclear ER membrane in yeast and plant cells. We further show that an equivalent helix in several plant- and human-infecting viruses of the alphavirus-like superfamily targets fluorescent proteins to the organelle membranes where they form their VRCs, including ER, vacuole, and Golgi membranes. Our work reveals a conserved helix that governs the localization of VRCs among a group of viruses and points to a possible target for developing broad-spectrum antiviral strategies.

Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection

SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.

SLC4A2 anion exchanger promotes tumour cell malignancy via enhancing net acid efflux across golgi membranes

Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, reverse their invasive and anchorage-independent growth phenotype. These findings suggest a possibility to return malignant cells to a benign state by restoring Golgi resting pH.

Closed-Loop Control between Two GTPase Switches makes the Secretory Functions of the Golgi Responsive to Growth Factors

Intercellular (between-cells) signals must be converted into an intracellular (within-cell) signal before it can trigger a proportionate response. How cells mount such proportionate responses within their interior remains unknown. Here we unravel the role of a coupled GTPase circuit on the Golgi membranes which enables the intracellular secretory machinery to respond proportionately to the growth factors in the extracellular space. The circuit, comprised of two species of biological switches, the Ras-superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase, Giαβγ, and their corresponding GAPs and GEFs, is coupled via at least one a forward and two key negative feedback loops. Interrogation of the circuit featuring such closed-loop control (CLC) using an integrated systems-based and experimental approach showed that CLC allows the two GTPases to mutually control each other and convert the expected switch-like behavior of Arf1 into an unexpected dose-response aligned (DoRA) linear behavior. Such behavior translates into growth factor-stimulated Giαβγ activity on Golgi membranes, temporal finiteness of Arf1 activity, and cellular secretion that is proportional to the stimuli. Findings reveal the importance of the coupled GTPase circuit in rendering concordant cellular responses via the faithful transmission of growth signals to the secretory machinery.

SLC4A2 Anion Exchanger Promotes Tumor Cell Malignancy via Enhancing H+ Leak across Golgi Membranes

Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes involves AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on both bicarbonate import (in exchange of chloride export) and the AE2a expression level in the cells. Imported bicarbonate anions and luminal protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The high surface-volume ratio of flattened Golgi cisternae helps this process, as their shape is optimal for water and gas exchange. Interestingly, this pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, to reverse cells’ invasive and anchorage-independent growth phenotype. These findings suggest that a malignant cell can be returned to a benign state by normalizing its Golgi resting pH.

Modeling the dynamic behaviors of the COPI vesicle formation regulators, the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1. This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used Fluorescence Recovery After Photobleaching data and kinetic Monte Carlo simulation to assess behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1, and suggests GBF1/E794K is stabilized on the membrane independently of complex formation.

Membrane targeting activates Leucine-rich repeat kinase 2 with differential effects on downstream Rab activation

ABSTRACTGenetic variation at the Leucine-rich repeat kinase 2 (LRRK2) locus contributes to risk of familial and sporadic Parkinson’s disease. Recent data have shown a robust association between localization to various membranes of the endolysosomal system and LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes are still poorly understood. Here we artificially direct LRRK2 to six different membranes within the endolysosomal system. We demonstrate that LRRK2 is activated and able to phosphorylate three of its Rab substrates (Rab10, Rab12 and Rab29) at each compartment. However, we report differing localization of pRab10 and pRab12 at the lysosomal and Golgi membranes. Specifically, we found that pRab10 colocalizes with a sub-population of perinuclear LRRK2-positive Golgi/lysosomal compartments whereas pRab12 localized to all LRRK2-positive Golgi/lysosomal membranes across the cell. When organelle positioning is manipulated by sequestering lysosomes to the perinuclear area, pRab10 colocalization with LRRK2 significantly increases. We also show recruitment of JIP4, a pRab10 effector that we have recently linked to LYTL, after trapping LRRK2 at various membranes. Taken together, we demonstrate that the association of LRRK2 to membranous compartments is sufficient for its activation and Rab phosphorylation independent of membrane identity. Our system also identifies a potential mechanism underlying the distinct relationships between LRRK2 and its substrates Rab10 and Rab12.

Abstract 13164: Tm6sf2 is a Regulator of Liver Fat Metabolism in Smooth Er Influencing Vldl Lipidation

Backgrounds: A variant in Transmembrane 6 Superfamily Member 2 [ TM6SF2 (E167K)] that is associated with a loss of function is strongly associated with both alcoholic and nonalcoholic fatty liver disease. TM6SF2 is a polytopic protein that is expressed predominantly in the liver and small intestines. Immunolocalization studies are consistent with the protein being present in the endoplasmic reticulum (ER) and the Golgi complex. Tm6sf2 -/- mice replicate the phenotype of individuals with TM6SF2 -167K variant: hepatic steatosis accompanied by hypocholesterolemia, and transaminitis. These mice have a reduced rate of secretion of VLDL-TG without any change in the rate of secretion of ApoB. Thus, TM6SF2 is required for normal lipidation of VLDL. To determine where in the secretory pathway TM6SF2 promotes lipid addition to nascent VLDL, we have generated Tm6sf2 -/- rats. Methods: Two lines of Tm6sf2 -/- rats with different frameshift mutations in exon 1 were generated using CRISPR/Cas9 technology. Rats were fasted 4 hours and tissues were collected and frozen at -80°C. Cell fractionation studies were performed to isolate the smooth and rough ER along with trans - and cis -Golgi membranes. Result: Both lines of rats were confirmed by immunoblotting to produce no TM6SF2 in the liver or intestines. The phenotype of the Tm6sf2 -/- rats resembles that of mice and humans. Cell fractionation studies revealed that TM6SF2 mainly localized to the smooth ER. Unlike what was observed previously by immunocytochemistry, no TM6SF2 was found in the Golgi fraction. Analysis of the lipid profile of the Golgi apparatus in the KO rat revealed that the majority triglyceride (TG) and fatty acids (FA) subclasses were decreased by one to two folds compared to WT in terms of TG or FA to ApoB48 ratios. Conclusions: TM6SF2 is localized predominantly in the smooth ER and regulates the lipidation of VLDL.

Wipi3 is essential for alternative autophagy and its loss causes neurodegeneration

Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.