DNAJB12 and Hsp70 Triage Arrested Intermediates of N1303K-CFTR for ER Associated-Autophagy

The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the ER's cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent soluble-state, and have a relatively long 3-hour half-life. ERAD-resistant pools of N1303K-CFTR are concentrated in ER-tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER-microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.

Cross-compartment signal propagation in the mitotic exit network

In budding yeast, the mitotic exit network (MEN), a GTPase signaling cascade, integrates spatial and temporal cues to promote exit from mitosis. This signal integration requires transmission of a signal generated on the cytoplasmic face of spindle pole bodies (SPBs; yeast equivalent of centrosomes) to the nucleolus, where the MEN effector protein Cdc14 resides. Here, we show that the MEN activating signal at SPBs is relayed to Cdc14 in the nucleolus through the dynamic localization of its terminal kinase complex Dbf2-Mob1. Cdc15, the protein kinase that activates Dbf2-Mob1 at SPBs, also regulates its nuclear access. Once in the nucleus, priming phosphorylation of Cfi1/Net1, the nucleolar anchor of Cdc14, by the Polo-like kinase Cdc5 targets Dbf2-Mob1 to the nucleolus. Nucleolar Dbf2-Mob1 then phosphorylates Cfi1/Net1 and Cdc14, activating Cdc14. The kinase-primed transmission of the MEN signal from the cytoplasm to the nucleolus exemplifies how signaling cascades can bridge distant inputs and responses.

DNAJB12 and Hsp70 Facilitate the Conformation Specific Degradation of Arrested N1303K-CFTR Intermediates by ER Associated-Autophagy

SUMMARYThe transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the ER’s cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is the second most common mutation in the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction bolding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent soluble-state, and have a relatively long 3-hour half-life. ERAD-resistant pools of N1303K-CFTR are concentrated in ER-tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and autolysosomes. Whereas, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exits site, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER-microdomains to triage in a conformation-specific manner membrane protein intermediates for secretion versus degradation via ERAD or selective-ER associated autophagy.

Cross-compartment signal propagation in the Mitotic Exit Network

ABSTRACTIn budding yeast, the Mitotic Exit Network (MEN), a GTPase signaling cascade integrates spatial and temporal cues to promote exit from mitosis. This signal integration requires transmission of a signal generated on the cytoplasmic face of spindle pole bodies (SPBs; yeast equivalent of centrosomes) to the nucleolus, where the MEN effector protein Cdc14 resides. Here, we show that the MEN activating signal at SPBs is relayed to Cdc14 in the nucleolus through the dynamic localization of its terminal kinase complex Dbf2-Mob1. Cdc15, the protein kinase that activates Dbf2-Mob1 at SPBs, also regulates its nuclear access. Once in the nucleus, priming phosphorylation of Cfi1/Net1, the nucleolar anchor of Cdc14, by the Polo-like kinase Cdc5 targets Dbf2-Mob1 to the nucleolus. Nucleolar Dbf2-Mob1 then phosphorylates Cfi1/Net1 and Cdc14, activating Cdc14. The kinase-primed transmission of the MEN signal from the cytoplasm to the nucleolus exemplifies how signaling cascades can bridge distant inputs and responses.

Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes

BackgroundAPOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs.MethodsImmunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies.ResultsBoth endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants.ConclusionsAPOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.

Complexity of the eukaryotic dolichol-linked oligosaccharide scramblase suggested by activity correlation profiling mass spectrometry

The canonical pathway of N-linked protein glycosylation in yeast and humans involves transfer of the oligosaccharide moiety from the glycolipid Glc3Man9GlcNAc2-PP-dolichol to select asparagine residues in proteins that have been translocated into the lumen of the endoplasmic reticulum (ER). Synthesis of Glc3Man9GlcNAc2-PP-dolichol occurs in two stages, producing first the key intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic face of the ER, followed by translocation of M5-DLO across the ER membrane to the luminal leaflet where the remaining glycosyltransfer reactions occur to complete the structure. Despite its critical importance for N-glycosylation, the scramblase protein that mediates the translocation of M5-DLO across the ER membrane has not been identified. Building on our ability to recapitulate scramblase activity in large unilamellar proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based ‘activity correlation profiling’ approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Curation of the activity correlation profiling data prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins were necessary for M5-DLO scramblase activity. Our results instead suggest the possibility that the M5-DLO scramblase may be a protein, or protein complex, whose activity is regulated at the level of quaternary structure. This key insight will aid future attempts to identify the scramblase.

Crystal structure of the N-terminal domain of the trypanosome flagellar protein BILBO1 reveals a ubiquitin fold with a long structured loop for protein binding

Trypanosoma brucei is a protist parasite causing sleeping sickness and nagana in sub-Saharan Africa. T. brucei has a single flagellum whose base contains a bulblike invagination of the plasma membrane called the flagellar pocket (FP). Around the neck of the FP on its cytoplasmic face is a structure called the flagellar pocket collar (FPC), which is essential for FP biogenesis. BILBO1 was the first characterized component of the FPC in trypanosomes. BILBO1's N-terminal domain (NTD) plays an essential role in T. brucei FPC biogenesis and is thus vital for the parasite's survival. Here, we report a 1.6-Å resolution crystal structure of TbBILBO1-NTD, which revealed a conserved horseshoe-like hydrophobic pocket formed by an unusually long loop. Results from mutagenesis experiments suggested that another FPC protein, FPC4, interacts with TbBILBO1 by mainly contacting its three conserved aromatic residues Trp-71, Tyr-87, and Phe-89 at the center of this pocket. Our findings disclose the binding site of TbFPC4 on TbBILBO1-NTD, which may provide a basis for rational drug design targeting BILBO1 to combat T. brucei infections.

Single-molecule imaging of the BAR-domain protein Pil1p reveals filament-end dynamics

Molecular assemblies can have highly heterogeneous dynamics within the cell, but the limitations of conventional fluorescence microscopy can mask nanometer-scale features. Here we adapt a single-molecule strategy to perform single-molecule recovery after photobleaching (SRAP) within dense macromolecular assemblies to reveal and characterize binding and unbinding dynamics within such assemblies. We applied this method to study the eisosome, a stable assembly of BAR-domain proteins on the cytoplasmic face of the plasma membrane in fungi. By fluorescently labeling only a small fraction of cellular Pil1p, the main eisosome BAR-domain protein in fission yeast, we visualized whole eisosomes and, after photobleaching, localized recruitment of new Pil1p molecules with ∼30-nm precision. Comparing our data to computer simulations, we show that Pil1p exchange occurs specifically at eisosome ends and not along their core, supporting a new model of the eisosome as a dynamic filament. This result is the first direct observation of any BAR-domain protein dynamics in vivo under physiological conditions consistent with the oligomeric filaments reported from in vitro experiments.