Assembly of the biogenesis of lysosome-related organelles complex-3 (BLOC-3) and its interaction with Rab9

Abstract

The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs.

FIGURE 1.

Hydrodynamic properties of BLOC-3. A, gel filtration profile of recombinant BLOC-3 on a Superose 6 column in 0.6% (w/v) β-OG. The hydrodynamic radii of standard proteins are indicated. B, Coomassie Blue-stained gel of purified BLOC-3 complex after gel filtration. Molecular masses are indicated in kDa. C, analytical ultracentrifugation of BLOC-3 at 4 °C in 0.6% (w/v) β-OG. Analysis using one species for the c(s) distribution results in a molecular mass of 146 kDa and an f/f₀ ratio of 1.65.

FIGURE 2.

Protease sensitivity and mapping of disordered regions. A, Sf9 cells were infected with either wild-type or a cathepsin/chitinase (v-cath/chiA)-deficient baculovirus expressing GST-HPS4 and HPS1. Total cell lysates taken at the indicated time points after infection were analyzed by immunoblotting using an antibody to GST. B, gel filtration profile of recombinant BLOC-3 after autoproteolytic digestion. The hydrodynamic radii of standard proteins are indicated. C, Coomassie Blue-stained SDS-PAGE of the peak fraction from the gel filtration shown in B. In addition to full-length HPS1, several smaller HPS1 and HPS4 fragments remain tightly associated in the complex (arrows). D, limited proteolysis with trypsin. Full-length recombinant BLOC-3 was incubated with 2 μg/ml trypsin at room temperature and aliquots taken at the indicated time points. E, BLOC-3 was proteolysed with trypsin for 15 min and loaded onto a Superdex 200 gel filtration column. The four major bands in the 15-min digest (D) remain tightly associated and co-elute in the peak fraction. However, the central region in HPS4 between 268 and 529 dissociates from the rest of the complex and is not present in the peak fraction. The gels in C through E were stained with Coomassie Blue, molecular masses are indicated in kDa. F, all protease cleavage sites in BLOC-3 are located in regions lacking predicted secondary structure. Mapped sites from autoproteolysis (open arrowheads) and limited trypsin proteolysis (closed) are shown together with predicted secondary structure elements for HPS1 and HPS4 (α-helices, red; β-strands, green).

FIGURE 3.

Assembly of the BLOC-3 complex. A, gel filtration profile of the BLOC-3 complex after removal of HPS4 residues 275–533 using flanking TEV sites. B, Coomassie Blue-stained peak fraction from the gel filtration shown in A. The N-terminal (1–274) and C-terminal (534–708) fragments of HPS4 remain tightly associated with HPS1, whereas the central region dissociates from the complex, showing that it is not needed for complex stability. C, N-terminal domains of HPS1 and HPS4 do not form a stable complex. GST-HPS4-(1–268) and HPS1-(1–248) were co-expressed, captured on glutathione resin, and detected by immunoblot using a rabbit polyclonal antibody against the full-length BLOC-3 complex. D, HPS4 N-terminal domain (1–218) interacts with full-length HPS1. Co-expression of GST-HPS4-(1–218) and full-length HPS1 followed by capture on glutathione resin and Coomassie Blue staining. Molecular masses in C and D are indicated in kDa. E, N-terminal domain of HPS4 forms soluble aggregates. Elution profile of HPS4-(1–218) on a Superdex 200 column after removal of the GST tag and ion exchange chromatography.

FIGURE 4.

GTP-dependent interaction of BLOC-3 with Rab9. A, Y2H assay of HPS1 and HPS4 binding to wild-type and GTP-locked (Q66L) Rab9a. B, Y2H assay of HPS4 binding to GTP-locked (Q66L) Rab9a and Rab9b. Negative and positive controls for interactions are described under “Experimental Procedures.” C, GST pulldown using purified recombinant BLOC-3 (200 nm) and immobilized wild-type GST-Rab9a (100 nm) loaded with either GDP or GMP-PNP. Coomassie Blue-stained gel. Molecular masses are indicated in kDa. D, GST pulldown using lysates from COS-7 cells expressing HA-HPS1, Myc-HPS4, or both. Lysates were passed over GST or GST-Rab9a loaded with GTP or GDP and bound proteins detected by immunoblotting with anti-HA and anti-Myc antibodies.

FIGURE 5.

Deleting the central region of HPS4 does not impair Rab9 binding. A, GST pulldown with different concentrations of recombinant BLOC-3 and GTP-locked GST-Rab9a at 100 nm. B, GST pulldown with different concentrations of recombinant BLOC-3(ΔHPS4_275–533) and GTP-locked GST-Rab9a at 100 nm. A GST contaminant is marked by an asterisk. Molecular masses in A and B are indicated in kDa. C, quantification of the titrations in A and B. The amount of bound BLOC-3 was determined by Coomassie Blue G250 fluorescence, normalized to the amount of GST-Rab9 in each lane and plotted against BLOC-3 concentration. Fitting the data assuming 1:1 binding stoichiometry results in a K_D of 82 nm for full-length BLOC-3 (blue) and 202 nm for BLOC-3(ΔHPS4_275–533) (red).

FIGURE 6.

The interaction of BLOC-3 with Rab9 is Rab-specific. A, GST pulldown using purified recombinant BLOC-3 (200 nm) and immobilized wild-type GST-Rab5a, -Rab7a, -Rab9a, and -Rab27a (100 nm) loaded with either GDP or GTPγS. Coomassie Blue-stained gel. Molecular masses are indicated in kDa. B, GST pulldown using mouse spleen cytosol and GST-Rab4a, -Rab7a and -Rab9a loaded with GTP or GDP as bait. Endogenous HPS4 protein was detected using an affinity-purified polyclonal antibody.

FIGURE 7.

Mapping the binding site on Rab9. A, scheme of Rab9-GMP-PNP in the active conformation (PDB 1YZL) with the residues mutated to alanine in B shown in stick representation. The switch I and II regions are colored yellow. Point mutations causing loss of HPS4 binding are highlighted in red and those with no effect in green. Deletion of the Val-109-Pro-112 loop (green backbone) also had no effect. B, Y2H assay of Rab9a(Q66L) binding to HPS4. Residues on the Rab9a surface were mutated to alanine as indicated. The ΔVKEP mutation (far right) refers to the Val-109—Pro-112 loop, which only occurs in Rab7, Rab9, Rab32, and Rab38.