A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu’s itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

In vivo reconstitution finds multivalent RNA-RNA interactions as drivers of mesh-like condensates

Liquid-like condensates have been thought to be sphere-like. Recently, various condensates with filamentous morphology have been observed in cells. One such condensate is the TIS granule network that shares a large surface area with the rough endoplasmic reticulum and is important for membrane protein trafficking. It has been unclear how condensates with mesh-like shapes, but dynamic protein components are formed. In vitro and in vivo reconstitution experiments revealed that the minimal components are a multivalent RNA-binding protein that concentrates RNAs that are able to form extensive intermolecular mRNA-mRNA interactions. mRNAs with large unstructured regions have a high propensity to form a pervasive intermolecular interaction network that acts as condensate skeleton. The underlying RNA matrix prevents full fusion of spherical liquid-like condensates, thus driving the formation of irregularly shaped membraneless organelles. The resulting large surface area may promote interactions at the condensate surface and at the interface with other organelles.

Acetyl-CoA flux from the cytosol to the ER regulates engagement and quality of the secretory pathway

Nε-lysine acetylation in the ER is an essential component of the quality control machinery. ER acetylation is ensured by a membrane transporter, AT-1/SLC33A1, which translocates cytosolic acetyl-CoA into the ER lumen, and two acetyltransferases, ATase1 and ATase2, which acetylate nascent polypeptides within the ER lumen. Dysfunctional AT-1, as caused by gene mutation or duplication events, results in severe disease phenotypes. Here, we used two models of AT-1 dysregulation to investigate dynamics of the secretory pathway: AT-1 sTg, a model of systemic AT-1 overexpression, and AT-1S113R/+, a model of AT-1 haploinsufficiency. The animals displayed reorganization of the ER, ERGIC, and Golgi apparatus. In particular, AT-1 sTg animals displayed a marked delay in Golgi-to-plasma membrane protein trafficking, significant alterations in Golgi-based N-glycan modification, and a marked expansion of the lysosomal network. Collectively our results indicate that AT-1 is essential to maintain proper organization and engagement of the secretory pathway.

Glucosylhydroxyceramides Modulate Secretion Machinery of a Subset of Plasmodesmata Proteins and a Change in the Callose Accumulation

The plasma membranes encapsulated in the plasmodesmata (PDs) with symplasmic nano-channels contain abundant lipid rafts, which are enriched by sphingolipids and sterols. The attenuation of sterol compositions has demonstrated the role played by lipid raft integrity in the intercellular trafficking of glycosylphosphatidylinositol (GPI)-anchored PD proteins, particularly affecting in the callose enhancement. The presence of callose at PD is tightly attributed to the callose metabolic enzymes, callose synthases (CalSs) and β-1,3-glucanases (BGs) in regulating callose accumulation and callose degradation, respectively. Sphingolipids have been implicated in signaling and membrane protein trafficking, however the underlying processes linking sphingolipid compositions to the control of symplasmic apertures remain unknown. A wide variety of sphingolipids in plants prompts us to investigate which sphingolipid molecules are important in regulating symplasmic apertures. Here, we demonstrate that perturbations of sphingolipid metabolism by introducing several potential sphingolipid (SL) pathway inhibitors and genetically modifying SL contents from two independent SL pathway mutants are able to modulate callose deposition to control symplasmic connectivity. Our data from pharmacological and genetic approaches show that the alteration in glucosylhydroxyceramides (GlcHCers) particularly disturb the secretory machinery for GPI-anchored PdBG2 protein, resulting in an over accumulated callose. Moreover, our results reveal that SL-enriched lipid rafts link symplasmic channeling to PD callose homeostasis by controlling the targeting of GPI-anchored PdBG2. This study elevates our understanding of the molecular linkage underlying intracellular trafficking and precise targeting to specific destination of GPI-anchored PD proteins incorporated with GlcHCers contents.

Structure and activation mechanism of the BBSome membrane protein trafficking complex

Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a key regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the β-propeller domain of BBS1, the subunit most frequently implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis results from folding defects and the disruption of autoinhibition and activation.