Multiple Rab GTPase binding sites in GCC185 suggest a model for vesicle tethering at the trans-Golgi

Abstract

GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether.

Figure 1.

Yeast two-hybrid analysis of Rab GTPase binding to distinct coiled-coils in GCC185. (A) Top, secondary structure prediction for GCC185 using Paircoils software. Residue number is shown at bottom. Middle, schematic representation of predicted coiled-coils: CC1, CC2, CC3, and fragments representing the remainder of the protein (ΔCC123, C343, and C110). Bottom, summary of Rab binding to each of the domains as determined by yeast two-hybrid analysis. Rab9A is asterisked because it was variably detected by two hybrid; biochemical experiments shown below confirm its binding to CC3. (B) Yeast two-hybrid data for the construct C343. Each box shows five colonies streaked on selective media; numbers represent activated versions of the human Rab proteins.

Figure 2.

Rab GTPase binding to purified GCC185 coiled-coils. (A) Coomassie stained SDS-polyacrylamide gel electrophoresis of untagged Rab proteins and GST-coiled-coil domains used in this study. Molecular mass is shown to the left. (B) Solution binding of [³⁵S]GTPγS-loaded Rab proteins to purified coiled-coil constructs. Reactions contained 3 μM coiled-coil and 0.1 μM active, nucleotide-loaded Rab protein. Products were collected on glutathione Sepharose and determined by scintillation counting. Binding to GST alone was subtracted; error bars represent SE of the mean for three independent experiments.

Figure 3.

(A) Helical wheel plot of residues near the C terminus of GCC185 CC3 predicts a solvent exposed leucine/isoleucine pair. (B) GCC185 I880A L873A are important for Rab9A binding to coiled-coil 3 (CC3). Binding was carried out as in Figure 2 by using the indicated concentrations of CC3 and 0.1 μM Rab9A.

Figure 4.

Coiled-coil 3 inhibits in vitro transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. (A) Ninety minute reactions were carried out as described in Reddy et al. (2006) and contained 10 μg of membranes, 0.5 mg/ml cytosol, and where indicated, 4 μM GST-tagged GCC185 coiled-coil domains. Reactions were corrected to the higher salt buffer of the coiled-coil preps added. Shown is a representative of three experiments carried out in triplicate; error bars represent SE of the mean. (B) In vitro transport in reactions containing GST-CC3. Values represent means of at least triplicates; shown are aggregate data from seven independent experiments.

Figure 5.

GCC185 coiled-coil deletion constructs containing an intact C terminus are correctly localized to the Golgi complex. Left column, myc-tagged constructs lacking the indicated domains (green); right column, Golgi localization as determined using rabbit anti-βCOP antibodies (red). Alexa488-goat anti mouse and Alexa594 goat anti-rabbit antibodies were used for detection. Bar, 10 μm.

Figure 6.

GCC185 coiled-coils one and two are required to rescue Golgi disruption caused by GCC185 depletion. (A) Cells were treated with fluorescein-labeled GCC185 siRNA for 72 h. After 48 h of siRNA treatment, cells were transfected with plasmids encoding the indicated myc-tagged proteins. Golgi localization was determined using mouse anti-GM130 and Cy5-goat anti-mouse antibodies (green); GCC185 rescue construct expression was detected using chicken anti-myc and Cy3-goat anti-chicken antibodies (red). Cells containing siRNA (as determined by detection of fluorescein) are indicated with asterisks. (B) Quantitation of rescue experiments. Golgi fragmentation was scored visually; data represent the average of two experiments in which a total of ≥40 cells were counted for each condition. Error bars represent SE of the mean of two independent experiments. Bar, 10 μm.

Figure 7.

Model for GCC185 function as a tether and Golgi organizer. (A) Dimensions of GCC185 if fully extended, transport vesicles and Golgi stacks. (B) A model for GCC185 in vesicle tethering. In this model, cytosolic GCC185 would first associate with Rab9-positive transport vesicles via GCC185 coiled-coil 3. GCC185 on vesicles brought close to the Golgi by cytoplasmic dynein could then engage Rab6 and Arl1 GTPases at the target membrane. SNARES would then drive fusion. (C) Although GCC185 is potentially long enough to reach across the stack, most molecules do not seem to; for details, see Figure 8 and text.

Figure 8.

Neither the N or C terminus of GCC185 colocalizes with the cis-Golgi marker GM130. (A) Confocal micrographs of cells either nontransfected (left column) or expressing N-terminally myc-tagged GCC185 (center column) or N-terminally myc tagged C110 (C-terminal) fragment (right column) were labeled for GM130 (green), endogenous GCC185 C terminus (left column only), or myc localization (center and right columns) as described in Materials and Methods. Bar, 10 μm. (B) Manders colocalization coefficients for each data set.