Fig 2: Proposed model of the VPS33B–VPS16B complex.A, top left, two copies of the AlphaFold predicted VPS33B structure (https://alphafold.ebi.ac.uk/entry/Q9H267) arranged in trans, consistent with our structural data (Fig. 6; C-terminal serine 617 highlighted in red). Bottom left, two copies of the AlphaFold VPS16B structure (https://alphafold.ebi.ac.uk/entry/Q9H9C1) with high-confidence region highlighted in green outline. Right, VPS33B–VPS16B complex 3D volume (gray) generated from negative-staining EM data (Fig. 5), above proposed model of the complex assembled from two VPS33B molecules bridged by three VPS16B. B, AlphaFold predicted structure of VPS16B with highlighted amino acids 143 to 316 (turquoise) we have identified as necessary for complex formation. C, AlphaFold predicted structure of VPS33B showing ARC-causing amino acid changes L30P, S243F, and H344D highlighted in red; the structures are rotated −90° along the Y-axis relative to each other. For AlphaFold predicted structures in (A) and (C), blue ribbon indicates per-residue confidence scores >70 (confident and very confident), and yellow ribbon indicates per-residue confidence scores <70 (low and very low confidence). ARC, arthrogryposis, renal dysfunction, and cholestasis; VPS, vacuolar protein sorting protein.

Fig 3: Negative-staining EM of VPS33B–VPS16B complex.A, representative micrograph of negative-stained glutaraldehyde-crosslinked purified VPS33B–VPS16B-3xFLAG complex; circles indicate representative particles used for 2D class averaging. Micrographs collected at 25,000× magnification with image pixel size of 2.61 Å; bar represents 200 nm. B, 2D class averages generated from 407 particles reveal an elongated two-lobed complex structure; bar represents 10 nm. VPS, vacuolar protein sorting protein.

Fig 4: Human VPS33B–VPS16B complex expressed in yeast is stable and well folded with a molecular weight of approximately 315 kDa.A, BN-PAGE immunoblots probed for VPS33B and VPS16B show the presence of both in human VPS33B–VPS16B-3xFLAG complexes affinity purified from yeast; major band comigrates with 480 kDa marker (arrowhead) as in HEK293 and DAMI cell lysates (Fig. 1A). B, Coomassie blue–stained SDS-PAGE gel indicates VPS33B and VPS16B are the only proteins present in purified complex, also shown in native form. C, on stained BN-PAGE gel. D, circular dichroism analysis of purified complex indicates mostly helical secondary structure. E, Representative SEC-MALS chromatogram for purified complex. F, SDS-PAGE immunoblots detected VPS33B and VPS16B in the major peak (fraction 2); results yielded a mean calculated molecular weight of 315 kDa for the complex (see Fig. S1 for light scattering data). BN-PAGE, blue native polyacrylamide gel electrophoresis; DAMI, megakaryoblastic DAMI cell line; HEK293, human embryonic kidney 293 cell line; VPS, vacuolar protein sorting protein.

Fig 5: The VPS33B–VPS16B complex contains multiple copies of each component.A–D, SDS-PAGE immunoblots show differentially tagged VPS33B and VPS16B expressed in transfected HEK293 cells can co-IP a second copy of each protein. A, probing for FLAG, HA, Myc, and GAPDH (bottom) of lysates from HEK293 cells cotransfected with HA-VPS33B + Myc-VPS16B, and with/without VPS16B-FLAG as indicated. B, in lysates from triple-transfected cells, anti-FLAG antibody co-IP’s HA-tagged VPS33B and Myc-tagged VPS16B, confirming at least two copies of VPS16B in the complex. C, probing of lysates from cells cotransfected with HA-VPS33B + Myc-VPS16B, and with/without VPS33B-FLAG. D, in lysates from triple-transfected cells, anti-FLAG antibody co-IP’s HA-tagged VPS33B and Myc-tagged VPS16B, confirming at least two copies of VPS33B in the complex. co-IP, coimmunoprecipitation; HA, hemagglutinin; HEK293, human embryonic kidney 293 cell line; VPS, vacuolar protein sorting protein.