The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases

Abstract

Originally identified in yeast, transport protein particle (TRAPP) complexes are Rab GTPase exchange factors that share a core set of subunits. TRAPPs were initially found to act on Ypt1, the yeast orthologue of Rab1, but recent studies have found that yeast TRAPPII can also activate the Rab11 orthologues Ypt31/32. Mammals have two TRAPP complexes, but their role is less clear, and they contain subunits that are not found in the yeast complexes but are essential for cell growth. To investigate TRAPP function in metazoans, we show that Drosophila melanogaster have two TRAPP complexes similar to those in mammals and that both activate Rab1, whereas one, TRAPPII, also activates Rab11. TRAPPII is not essential but becomes so in the absence of the gene parcas that encodes the Drosophila orthologue of the SH3BP5 family of Rab11 guanine nucleotide exchange factors (GEFs). Thus, in metazoans, Rab1 activation requires TRAPP subunits not found in yeast, and Rab11 activation is shared by TRAPPII and an unrelated GEF that is metazoan specific.

Figure 1.

The Rab GEFs of the Drosophila Golgi apparatus. (A) Cartoon of the SBP-GFP TAP tag and the strategy used to make stable cell lines or fly lines by CRISPR and then analyzing the tagged proteins in cell lines or flies. AC, affinity chromatography; MS/MS, tandem mass spectrometry. (B) Widefield micrographs of cells transiently transfected with TAP-tagged Golgi GEFs and labeled with antibodies to the indicated Golgi markers. Images are representative of at least two independent experiments, with at least three micrographs obtained from each. Bars, 5 µm. (C) Immunoblot of stable cell lines expressing SBP-GFP–tagged TRAPPC3 or Rgp1. (D) TAP of TRAPPC3 from stably transfected cell lines. The purification was on anti-GFP beads with elution by TEV protease and then on streptavidin beads with elution by biotin. The starting tagged protein (arrows) and the final purified protein (arrowhead) are indicated. FT, flowthrough. (E) Silver-stained protein gel comparing single-step affinity chromatography of TRAPPC3–SBP-GFP using the GFP tag (TEV eluate) with TAP using both the GFP and the SBP tag (biotin eluate). Molecular masses are given in kilodaltons. (F) Mass spectrometry analysis of TAPs of SBP-GFP–tagged TRAPPC3 and Rgp1. For each protein that was identified by mass spectrometry, the total spectral counts are shown for the TRAPPC3 or Rgp1 purifications. Duplicates are shown. CG, computed gene; Cont., untransfected cells.

Figure 2.

The TRAPP complexes of Drosophila. (A) Mass spectrometry analysis of TRAPPC3, TRAPPC9, TRAPPC10, TRAPPC11, TRAPPC12, and TRAPPC13 tandem affinity purified from stably transfected cell lines. Total spectral counts for each protein are shown, with replicates shown in Fig. S1 B. Precipitations also contained lower levels of abundant chaperones and cytosolic enzymes. With the shared TRAPP subunit TRAPPC3, only three proteins apart from known TRAPP subunits were consistently present across all TAP purifications and several single-step purifications (catalase, ribosomal protein S26, and Gdi), and these were only present at trace levels in the TAP samples and so were not pursued further. (B) Immunoblot using the TRAPPC9 and TRAPPC12 antibodies of proteins associated with Rgp1, TRAPPC3, TRAPPC8, TRAPPC10, TRAPPC11, and TRAPPC13 after anti-GFP affinity purification from lysates of transiently transfected S2 cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation. (C) Subunits present in the different the TRAPP complexes of yeast, flies, and humans. (D) Widefield micrographs of S2 cells transiently expressing GFP-tagged TRAPPC8, TRAPPC11, and TRAPPC13 and stained with antibodies against Golgin245 and GM130. Line scans of fluorescence intensity relative to the maximum are from representative Golgi stacks (dotted line on Golgin245 panels). The TRAPP subunits are enriched at the cis end of the stack. (E) Confocal micrographs of S2 cells labeled with antisera to endogenous TRAPPC9 (TRAPPII) or TRAPPC12 (TRAPPIII) and the Golgi proteins Golgin245 or GMAP. Line scans are from representative Golgi stacks (dotted line on Golgi marker panels). Bars, 5 µm. Images in D and E are representative of at least two independent experiments, with at least three micrographs obtained from each.

Figure 3.

TRAPPII and TRAPPIII are present in embryos, larvae, and adults. (A) Silver-stained protein gels of TAPs of SBP-GFP–tagged TRAPPC11, TRAPPC9, TRAPPC3, and TRAPPC12 from embryos. (B) Mass spectrometry analysis of proteins associated with SBP-GFP–tagged TRAPPC11, TRAPPC9, TRAPPC3, and TRAPPC12 after TAP from embryos. Total spectral counts for each protein are shown. (C) Immunoblot analysis using the TRAPPC9 and TRAPPC12 antibodies of proteins associated with TRAPPC3, TRAPPC9, TRAPPC11, and TRAPPC12 after anti-GFP immunoprecipitation (IP) from lysates prepared from adults (left) or wandering third instar larvae (right) carrying SBP-GFP–tagged alleles. Arrows indicate GFP-tagged TRAPP subunits; arrowheads indicate native TRAPP subunits. Molecular masses are given in kilodaltons. (D) Confocal micrographs of SBP-GFP–tagged TRAPPC3, TRAPPC11, and TRAPPC12 in wing imaginal discs and salivary glands dissected from wandering third instar larvae and stained for GFP, GM130, and Golgin245. In each case, the tag was inserted at the genomic locus, and therefore, the tagged protein was expressed from its native promoter. Line scans are from representative Golgi stacks (dotted lines in GFP/Golgin245 panels). In the salivary gland, all three proteins were discretely located to both the cis and trans ends of the stack (arrows in insets), with a similar bimodal distribution also observed in follicle cells (not depicted). Bars: (main images) 5 µm; (insets) 1 µm. Images are representative of at least two independent experiments, with at least three micrographs obtained from each.

Figure 4.

TRAPPII is required for male fertility, and TRAPPIII is essential for viability. (A) Summary of mutations generated in TRAPP subunits in this and previous studies. The nature of the lesions in the EMS alleles of TRAPPC2, TRAPPC8, and TRAPPC11 were determined by sequencing the locus from the relevant mutants (Fig. S2 B). (B) Mass spectrometry analysis of TRAPPC9–SBP-GFP tandem affinity–purified from a WT or TRAPPC10[16] mutant background. Total spectral counts for each protein are shown. Subunits with a thick line in the cartoon are essential for Rab1 nucleotide exchange activity in yeast. (C) TRAPPC11 is essential for adult (blue bars) and pupal (green bars) viability. The bars show mean numbers of adult or pupal progenies calculated from two parallel crosses (pupal lethality of TRAPPC11[MB06920]/deficiency heterozygotes and TRAPPC11[63BD2]/deficiency heterozygotes) or three parallel crosses (all other heterozygotes). The mean value is shown above each bar. Note that TRAPPC11[63BD2]/deficiency heterozygotes occasionally develop into pupae, suggesting the allele is a strong hypomorph. (D) TRAPPC9 and TRAPPC10 are not essential for adult viability. The bars show the mean number of adult progeny obtained in three independent crosses. (E) TRAPP9 and TRAPPC10 are required for male fertility. The bars show the mean number of pupal progeny from three independent crosses using males with the indicated allele over the relevant deficiency. Error bars show SD.

Figure 5.

TRAPPII complex activates the Rab GTPases Rab1 and Rab11, whereas TRAPPIII only shows GEF activity toward Rab1. (A) Coomassie blue–stained protein gels of recombinant Drosophila TRAPP complexes purified from Sf9 cells coexpressing the subunits of each complex. FLAG tags on C10 or C11 allowed isolation of TRAPPII or TRAPPIII, respectively. PreScission protease (GST-HRV-3C protease) was used to cleave the tags (asterisks) and was subsequently removed using glutathione Sepharose beads. C10 also copurified with C9 in the absence of the shared subunits (TRAPPII lane). The Hsc70 chaperone (CG4264) is a contaminant of the TRAPPII purification protocol. Molecular masses are given in kilodaltons. (B) Release of mant-GDP from 250 nM of Rab-His₆ by 50 nM of TRAPPII or TRAPPIII in the presence or absence of synthetic fly Golgi mix liposomes. Traces are the mean of at least three experiments. Error bars show SEM.

Figure 6.

TRAPPIII is required for cell growth and Rab1 localization. (A) Widefield micrographs of wing imaginal discs in which mitotic clones had been induced using the Flp-FRT system 2.33 or 3 d before fixation. This method generates homozygous clones of cells in a heterozygous background. The control clones or clones mutant for TRAPPC3[77] or TRAPPC11[7] are marked by the absence of RFP-tagged histone that is expressed from the chromosome that carries the mutation (arrows in inset zoom). Clones lacking TRAPPC3 or TRAPPC11 are only detectable at the earlier time point. Bars: (main images) 100 µm; (insets) 20 µm. (B and C) Confocal micrographs of YFP-Rab1 in clones of cells mutant for TRAPPC11[7] or TRAPPC3[77] in the peripodial cells of the wing imaginal discs costained for YFP and the Golgi markers GMAP or Golgin245 or the ER exit site marker Sec16. Mutant clones are marked by loss of RFP-tagged histone, with the nuclei stained with DAPI. Loss of TRAPPC11 or TRAPPC3 reduces YFP-Rab1 on the Golgi without affecting the other markers. Bars: (main images) 10 µm; (insets) 2 µm. Images are representative of at least two independent experiments, with at least three micrographs obtained from each. (D) Comparison of the levels on the Golgi of YFP-Rab1 and of Golgi/ER exit site markers in the homozygous mutant clones for TRAPPC3^−/− versus the surrounding WT tissue. Fluorescent intensities were extracted from micrographs such as those shown in C, and in each case, n = 20 Golgi from two wing discs. Mean values are shown. Error bars show SD. The level of YFP-Rab1 showed a statistically significant reduction in the mutant clones, whereas the other markers were not significantly affected (two-tailed nonparametric Mann-Whitney test).

Figure 7.

Parcas and TRAPPII are both Rab11 GEFs and together serve an essential role in Drosophila. (A) Release of mant-GDP from 250 nM of Rab-His₆ by 50 nM of Parcas in the presence or absence of synthetic fly Golgi mix liposomes (Lipo). The Rabex domain of the Rab5 GEF Vps9 (50 nM) was used as a negative control. Traces are means. (B) Rab11 nucleotide exchange rates calculated from the plots in A. Values are means ± SEM (Parcas, n = 3; Vps9, n = 2). (C) Nucleotide exchange rates of Parcas and the Rabex domain of Vps9 on the indicated Rabs. Parcas acts on Rab11 but not Rab1 or Rab5. (D) The mean number of adult progeny with homozygous or heterozygous genotypes obtained from three independent crosses, each using five females and three males carrying the indicated mutations. The independent values are shown along with the mean. Error bars show SD. Flies were viable if they lacked Parcas or the TRAPPII subunit TRAPPC9, but not both.