BORC assemblies integrate BLOC-1 subunits to diversify endosomal trafficking functions

Abstract

BORC and BLOC-1 are multisubunit complexes that regulate endolysosomal trafficking. Although they are presumed to be distinct, their paralogous origins and shared subunits suggest the potential for higher-order assembly. Here, we reveal the conserved octameric architecture of BORC formed by two intertwined tetramers and present the structure of C. elegans BORC. Through cross-linking mass spectrometry of endogenous complexes, we validate this model for human BORC and demonstrate that the integrity of the complex, which is essential for lysosomal transport, relies on specific interfacial residues. We also clarify the disruptive nature of disease-causing mutations and propose that the formation and function of BORC are likely regulated by specific cues. These cues might include the phosphorylation of Snapin and a pH-sensitive histidine residue in BORCS5. Additionally, we present direct biochemical and structural evidence of BORC-BLOC-1 hybrid complexes. Finally, we link a specific hybrid complex to the regulation of transferrin receptor recycling via interaction with the EARP complex. Our work challenges the paradigm of BORC and BLOC-1 as separate entities, establishing a model of dynamic complex formation wherein modular assembly creates functional specialization to meet diverse cellular demands.

Keywords: BLOC-1; BORC; EARP; lysosome; recycling endosome.

Fig. 1.

Integrative structure-biology approach reveals the molecular basis of BORC complex formation. (A) Distances between Cα atoms observed for the detected cross-linked peptides in the Alphalink2 model. Left side, results for all cross-links with a score >100, Right hand side, same analysis excluding cross-links in unstructured termini. (B) Side view of the superposition of the AlphaFold3 and the AlphaLink2 models of C. elegans BORC. In the AlphaFold3, BORCS5 is represented in dark olive green, BORCS6 in orange, BORCS7 in yellow green, BORCS8 in saddle brown, KxD1 in dark goldenrod, Snapin in dark cyan, BLOC1S1 in medium aquamarine and finally BLOC1S2 in dark slate gray. All subunits from the Alphalink2 model are shown in dark blue. The RMSD of the analysis is indicated. (C) AlphaLink2 model fitted into the CryoEM density. BORCS5 is presented in dark olive green, BORCS6 in orange, BORCS7 in yellow green, BORCS8 in saddle brown, KxD1 in dark goldenrod, Snapin in dark cyan, BLOC1S1 in medium aquamarine and finally BLOC1S2 in dark slate gray. (D) CryoEM density fitted C. elegans BORC model with indicated cross-links with a score >100 in blue.

Fig. 2.

In vivo functional validation of BORC’s structure. (A) Expression of SH-tagged BORCS6 wild type was induced by incubating the corresponding HEK293 Flp-In T-REx cell lines with 20 ng/mL tetracycline. Streptavidin affinity precipitates and respective inputs were analyzed by immunoblotting. SH, StrepII-HA-tag; HA, hemagglutinin. n = 2 independent biological experiments. (B–E) Experiments were performed as described in (A) for BORCS7 (n = 3), BORCS5 (n = 3), Snapin (n = 3), and BORCS8 (n = 2) respectively, (F) Quantification of the experimental data from panels (B–D). (G) Indirect immunofluorescence images of HeLa wild type, BORCS7 KO, and reconstituted cell lines kept under starved or stimulated conditions. Merged and single-channel images of endogenous lysosomal marker LAMP1 (red) and Phalloidin (green) are indicated. Representative cells are shown. n = 3 independent biological experiments. (Scale bar, 10 μm.) (H) Indirect immunofluorescence images of HeLa wild type, BORCS5 KO, and reconstituted cell lines kept under starved or stimulated conditions. Merged and single-channel images of endogenous lysosomal marker LAMP1 (red) are indicated. Representative cells are shown. n = 3 independent biological experiments. (Scale bar, 10 μm.) (I) The lysosomal distribution of HeLa wild type, BORCS7KO, and reconstituted lines was analyzed based on the experiment shown in (G), Graphic depicts the distance of each lysosome to the nucleus in µm. The vertical black line represents the median, the dotted line shows the 17 µm distance from the nucleus distinguishing peripheral from perinuclear lysosomes. A minimum of 19 cells were analyzed with more than 5,235 lysosomes per condition. (J) The lysosomal distributions of HeLa wild type, BORCS5KO, and reconstituted lines were analyzed based on the experiment shown in (H) Graphic depicts the distance of each lysosome to the nucleus in µm. The vertical black line represents the median, the dotted line shows the 17 µm distance from the nucleus distinguishing peripheral from perinuclear lysosomes. A minimum of 18 cells were analyzed with more than 4,710 lysosomes per condition.

Fig. 3.

Monitoring the existence of BORC/BLOC-1 mixed complexes. (A) HEK293 Flp-In T-REx cell lines induced to trigger the expression of SH-tagged BORCS7wt, BORCS7_1-88, SH-tagged BORCS5wt, and respective mutants or SH.GFP, were either starved for FBS or stimulated. The eluates from Streptavidin affinity precipitations were subjected to mass spectrometry analysis. Each graphic displays the comparison between the interactome of the mutant mentioned in the center of the scheme and the respective wt control. The copurified subunits are ranked clockwise by their abundance in the WT interactome. The ratio of the abundances of each interactor in the mutant vs wt interactomes is shown in a color gradient from red to dark blue. The statistical significance of the abundance ratio is indicated by the line surrounding the circle. BLOC-1 specific components are encircled in yellow. The components of BORC present in the opposite tetramer of the complex relative to the bait are encircled with a green line, n = 3 independent biological experiments. (B) HA.BORCS7 was stably expressed in the background of the BORCS7KO, BORCS6KO, or BORCS5KO. All cell lines were subjected to anti-HA immunoprecipitation under steady state conditions. Eluates and respective inputs were analyzed by immunoblotting. HA, hemagglutinin. n = 4 independent biological experiments. (C) Mass spectrometry analysis of the eluates obtained in (B). Graphic representation follows the same pictogram rules as in panel (A) with the exception that the abundance values of HA.BORCS7 are also shown. (D) In vitro affinity purification of BORC hexamers. SDS-PAGE gel was stained with coomassie. (E) HEK293 Flp-In T-REx cell lines inducibly expressing BORC components or BLOC1S6 were subjected to Streptavidin affinity precipitation. Eluates composition was analyzed by mass spectrometry. The table represents the abundance of each subunit in the different interactomes, normalized to the levels found using Snapin as a bait. n = 4 independent biological experiments. (F) Scheme representing the putative composition of mixed complexes A and B.

Fig. 4.

Existence of mixed complex A and possible physiological function of a mixed complex assembly. (A) AlphaFold3 prediction of mixed complex A (iPTM 0.46). BORCS6 is represented in orange, BLOC1S1 in medium aquamarine, BORCS8 in saddle brown, KxD1 in dark goldenrod, BLOC1S2 in dark slate gray, Snapin in dark cyan, BLOC1S5 in tomato and DTNBP1 in maroon. The structure is supported by a total of 11 cross-links (<5 % FDR) detected in SH-BORCS6 AP-XL-MS experiments. Cross-links between BORC exclusive subunits are depicted in dashed lines. Solid lines represent cross-links between BORC and BLOC-1 subunits with those under 25 Å Cα–Cα distance in red and those above 25 Å in blue. (B) Zoom view of the central region of mixed complex A depicted in the previous panel. (C) Cross-links between BORC and BLOC-1 components exceeding Cα–Cα distances of 25 Å are represented in black and connect flexible terminal residues of DTNBP1 and BLOC1S5 to the core of the helix bundle. Structured rigid regions (pLDDT > 59) are colored in blue and flexible or disordered regions (pLDDT < 59) in red. (D) Schematic representation of the cross-links between EARP subunits and BORC/BLOC-1 components found in the SH-BORCS6 and BORCS7SH AP-XL-MS experiments. Line thickness correlates with the number of cross-links. (E) Schematic representation of the transferrin recycling experiment, (F) Indirect immunofluorescence images of HeLa wild type, BORCS5KO, BORCS6KO, and BORCS7KO cells at different time points post internalization of labeled transferrin. Merged and single-channel images of transferrin-Alexa488 (green) and EEA1 are indicated. Representative cells are shown. n = 3. (Scale bar, 10 μm.) (G) Graphic depicts the colocalization rate between labeled transferrin and endogenous EEA1 in the different cell lines, 10 cells per genotype were analyzed, (H) Graphical representation of the levels of transferrin-Alexa488 found in the cells normalized to the levels of labeled transferrin at time point 0 of Wild type HeLa, 10 cells per genotype were analyzed. (I) Graphical representation of the putative role of mixed BORC/BLOC-1 complexes in transferrin recycling.