Solution Structure of Human Pex5��Pex14��PTS1 Protein Complexes Obtained by Small Angle X-ray Scattering

The Pex5p receptor recognizes newly synthesized peroxisomal matrix proteins which have a C-terminal peroxisomal targeting signal to the peroxisome. After docking to protein complexes on the membrane, these proteins are translocated across the membrane. The docking mechanism remains unclear, as no structural data on the multicomponent docking complex are available. As the interaction of the cargo-loaded Pex5p receptor and the peroxisomal membrane protein Pex14p is the essential primary docking step, we have investigated the solution structure of these complexes by small angle x-ray scattering and static light scattering. Titration studies yielded a 1:6 stoichiometry for the Pex5p·Pex14p complex, and low resolution structural models were reconstructed from the x-ray scattering data. The free full-length human Pex5p is monomeric in solution, with an elongated, partially unfolded N-terminal domain. The model of the complex reveals that the N terminus of Pex5p remains extended in the presence of cargo and Pex14p, the latter proteins being significantly intermingled with the Pex5p moiety. These results suggest that the extended structure of Pex5p may play a role in interactions with other substrates such as lipids and membrane proteins during the formation of functional multiprotein complexes.Peroxisomes are ubiquitous organelles in eukaryotes which are involved in different metabolic pathways (1). Peroxisomal matrix proteins, which contain a peroxisomal targeting signal (PTS),⁴ are imported into the peroxisome by recognition of two different import receptors, Pex5p or Pex7p. These receptors recognize specific signal sequences, PTS1 and PTS2, respectively (1). At the molecular level the C-terminal PTS1 signal is bound in a central cavity of the ring-like structure of the seven tetrapeptide repeat (TPR) domains of the C-terminal part of Pex5p (Pex5p(C)) (2–5). It was recently proposed that some of the structural principles of the Pex5p/cargo interaction may also apply to the PTS2 cargo recognition of the Pex7p receptor (5).The next step of PTS-protein import, docking of the cargo loaded receptor to the translocon, involves the peroxisomal protein Pex14p (6). Multiple Pex14p binding sites with di-aromatic pentapeptide motifs (WXXX(F/Y)) were shown to be present in the N terminus of Pex5p (7–9). The number of these motifs, however, varies among species. The human Pex5p receptor, which has been investigated in this contribution, has a total of seven motifs. A recent NMR structure of the N-terminal domain of Pex14p and the first WXXX(F/Y) motif of Pex5p reveals an α-helical conformation of the motif (10). Interactions between Pex5p and other proteins and by their association with the peroxisomal membrane possibly lead to dissociation of the PTS-protein from Pex5p (11–13). The exact sequence of events in the import mechanism remains, however, unknown. It is in particular unclear how, in contrast with other organelles, peroxisomes can import folded oligomeric, functional proteins (14).Previous biophysical work indicated that the N terminus half of Pex5p is unfolded in vitro (15, 16). Recent protease sensitivity assays showed that the proteolytic profiles of the full-length receptor Pex5p(F) change in the presence of PTS1 peptide and the Pex13p Src homology 3 domain, which is another docking factor (16, 17), indicating conformational changes of Pex5p upon binding these receptor ligands. Furthermore, it was found that Pex5p may even traverse the peroxisomal membrane, leaving only a small N-terminal fragment in the cytosol while exposing the C-terminal TPR domain to the luminal side of the membrane (11).Although recognition of many PTS cargos seems to be confined to the C-terminal TPR domains of Pex5p, it has become clear that the N-terminal part of Pex5p is primarily involved in docking of the receptor onto the peroxisomal membrane and other docking factors. Because only poorly diffracting crystals have been purified to date, we investigated its solution structure by small angle x-ray scattering (SAXS) and static light scattering (SLS). Complexes with the PTS1 cargo sterol carrier protein 2 (SCP2), which functions as lipid transfer protein, were also studied as the crystal structure of Pex5p(C)/SCP2 is already known (4). Our results indicate that human Pex5p(F) is a monomer with an extended N terminus. The stoichiometry of Pex5p(F)·Pex14p(N)·PTS1 complex has been assessed by titration with SAXS, SLS, and gel filtration, and a low resolution structural model of the complex has been reconstructed in which Pex5p(F) remains extended upon Pex14p(N) binding.     

Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components

Most eukaryotic cells require peroxisomes, organelles housing fatty acid β-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired β-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.Oilseed plants obtain energy for germination and early development by utilizing stored fatty acids (Graham, 2008). This β-oxidation of fatty acids to acetyl-CoA occurs in peroxisomes, organelles that also house other important metabolic reactions, including the glyoxylate cycle, several steps in photorespiration, and phytohormone production (Hu et al., 2012). For example, indole-3-butyric acid (IBA) is β-oxidized into the active auxin indole-3-acetic acid (IAA) in peroxisomes (Zolman et al., 2000, 2007, 2008; Strader et al., 2010; Strader and Bartel, 2011). Many peroxisomal metabolic pathways generate reactive oxygen species (Inestrosa et al., 1979; Hu et al., 2012), and peroxisomes also house antioxidative enzymes, like catalase and ascorbate peroxidase, to detoxify hydrogen peroxide (Wang et al., 1999; Mhamdi et al., 2012).Peroxisomes can divide by fission or be synthesized de novo from the endoplasmic reticulum (ER). Preperoxisomes with peroxisomal membrane proteins bud from the ER and fuse, allowing matrix proteins to be imported to form mature peroxisomes (van der Zand et al., 2012; Mayerhofer, 2016). Peroxin (PEX) proteins facilitate peroxisome biogenesis and matrix protein import. Most peroxins are involved in importing proteins destined for the peroxisome matrix, which are imported after recognition of a type 1 or type 2 peroxisome-targeting signal (PTS). The PTS1 is a tripeptide located at the C terminus of most peroxisome-bound proteins (Gould et al., 1989; Chowdhary et al., 2012). The less common PTS2 is a nonapeptide usually located near the N terminus (Swinkels et al., 1991; Reumann, 2004). PTS1 proteins are recognized by PEX5 (van der Leij et al., 1993; Zolman et al., 2000), PTS2 proteins are recognized by PEX7 (Marzioch et al., 1994; Braverman et al., 1997; Woodward and Bartel, 2005), and PEX7 binds to PEX5 to allow matrix protein delivery in plants and mammals (Otera et al., 1998; Hayashi et al., 2005; Woodward and Bartel, 2005). The cargo-receptor complex docks with the membrane peroxins PEX13 and PEX14 (Urquhart et al., 2000; Otera et al., 2002; Woodward et al., 2014), and PEX5 assists cargo translocation into the peroxisomal matrix (Meinecke et al., 2010) before dissociating from its cargo (Freitas et al., 2011).After cargo delivery, PEX5 is recycled to enable further rounds of cargo recruitment (Thoms and Erdmann, 2006). This process requires a set of peroxins that is implicated in ubiquitinating PEX5 so that it can be retrotranslocated back to the cytosol. PEX5 ubiquitination is best understood in yeast. In Saccharomyces cerevisiae, Pex5 is monoubiquitinated through the action of the peroxisome-tethered ubiquitin-conjugating enzyme Pex4 and the peroxisomal ubiquitin-protein ligase Pex12 (Platta et al., 2009) and returned to the cytosol with the assistance of a peroxisome-tethered ATPase complex containing Pex1 and Pex6 (Grimm et al., 2012). S. cerevisiae Pex5 also can be polyubiquitinated and targeted for proteasomal degradation (Kiel et al., 2005). The cytosolic ubiquitin-conjugating enzyme Ubc4 cooperates with the peroxisomal ubiquitin-protein ligase Pex2 to polyubiquitinate Pex5 (Platta et al., 2009). Pex10 has ubiquitin-protein ligase activity (Williams et al., 2008; Platta et al., 2009; El Magraoui et al., 2012), but whether Pex10 directly ubiquitinates Pex5 is controversial. Pex10 promotes Ubc4-dependent Pex5 polyubiquitination when Pex4 is absent (Williams et al., 2008); however, Pex10 is not essential for Pex5 mono- or polyubiquitination (Platta et al., 2009), but rather enhances both Pex4/Pex12- and Ubc4/Pex2-mediated ubiquitination (El Magraoui et al., 2012). Recycling of the PTS2 receptor PEX7 is less understood, although the Pex5 recycling pathways are implicated in shuttling and degrading Pex7 in Pichia pastoris (Hagstrom et al., 2014).Although PEX5 ubiquitination has not been directly demonstrated in plants, the implicated peroxins are conserved in Arabidopsis, and several have been connected to PEX5 retrotranslocation. The PEX4 ubiquitin-conjugating enzyme binds to PEX22, which is predicted to be a peroxisomal membrane protein based on ability to restore peroxisome function to yeast mutants (Zolman et al., 2005). The pex4-1 mutant displays increased membrane-associated PEX5 (Ratzel et al., 2011; Kao and Bartel, 2015), suggesting that ubiquitin supplied by PEX4 promotes PEX5 retrotranslocation. PEX1 and PEX6 are members of the ATPases associated with diverse cellular activities (AAA) family and are tethered to peroxisomes by the peroxisomal membrane protein PEX26 (Goto et al., 2011; Li et al., 2014). The pex6-1 mutant displays PTS1 import defects and decreased PEX5 levels (Zolman and Bartel, 2004), suggesting that impaired PEX5 recycling can lead to increased PEX5 degradation. Indeed, pex4-1 restores PEX5 levels in the pex6-1 mutant (Ratzel et al., 2011), suggesting that Arabidopsis PEX4 also is involved in PEX5 ubiquitination and degradation when retrotranslocation is impeded.In addition to allowing for further rounds of PTS1 cargo import, several lines of evidence suggest that in the absence of efficient retrotranslocation, PEX5 retention in the peroxisomal membrane impairs peroxisome function. Slightly reducing levels of the PEX13 docking peroxin ameliorates the physiological defects of pex4-1 without restoring matrix protein import (Ratzel et al., 2011), presumably because decreasing PEX5 docking reduces its accumulation in the peroxisomal membrane. In addition, overexpressing PEX5 exacerbates rather than ameliorates the peroxisomal defects of pex4-1 (Kao and Bartel, 2015), suggesting that pex4-1 defects are linked to excessive PEX5 lingering in the peroxisome membrane rather than a lack of PEX5 available for import.The three Really Interesting New Gene (RING) peroxins (PEX2, PEX10, and PEX12) from Arabidopsis each possesses in vitro ubiquitin-protein ligase activity (Kaur et al., 2013). Null mutations in the RING peroxin genes confer embryo lethality in Arabidopsis (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Fan et al., 2005; Prestele et al., 2010), necessitating other approaches to study the in vivo functions of these peroxins. Expressing RING peroxins with mutations in the C-terminal zinc-binding RING domains (ΔZn) confers matrix protein import defects for PEX2-ΔZn and photorespiration defects for PEX10-ΔZn but no apparent defects for PEX12-ΔZn (Prestele et al., 2010). Targeting individual RING peroxins using RNAi confers β-oxidation deficiencies and impairs PTS1 cargo import (Fan et al., 2005; Nito et al., 2007). A screen for delayed matrix protein degradation (Burkhart et al., 2013) uncovered a missense pex2-1 mutant and a splicing pex10-2 mutant that both display PTS1 import defects (Burkhart et al., 2014), suggesting roles in regulating the PTS1 receptor, PEX5. A missense pex12 mutant (aberrant peroxisome morphology 4, apm4) has defects in β-oxidation and PTS1 import and increased membrane-associated PEX5 (Mano et al., 2006). These findings highlight the essential roles of the RING peroxins in Arabidopsis development and peroxisomal functions, but the RING peroxin interactions and the individual roles of the RING peroxins in PEX5 retrotranslocation remain incompletely understood.In this study, we describe a missense pex12-1 mutant recovered from a forward genetic screen for β-oxidation deficient mutants. The pex12-1 mutant displayed severe peroxisomal defects, including reduced growth, β-oxidation deficiencies, matrix protein import defects, and inefficient processing of PTS2 proteins. Comparing single and double mutants with impaired RING peroxins revealed that each RING peroxin contributes to complex stability and influences PEX5 accumulation. Furthermore, decreasing PEX4 function ameliorated pex12-1 defects, suggesting that the Glu-to-Lys substitution in pex12-1 lures ubiquitination, perhaps by pex12-1 itself, leading to PEX4-dependent degradation of the mutant protein.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection

The cell''s endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system.Membrane compartmentalization is an essential mechanism for eukaryotic life, by which cells separate and control biological processes. Plant growth, development, and adaptation to biotic and abiotic stress all rely on the highly dynamic endomembrane system, yet we know comparatively little about the proteins regulating these dynamic trafficking events. The plasma membrane (PM) provides the main interface between the cell and its environment, mediating the transfer of material to and from the cell and is a primary site for perception of external signals. Transmembrane proteins are synthesized in the endoplasmic reticulum (ER) and trafficked to the PM via the Golgi, although there are other secretory routes for soluble cargo (discussed in (1–4)). Post-Golgi trafficking is the main route by which newly synthesized transmembrane proteins and cell wall glycans are delivered to the PM. In plants, secretory and endocytic traffic converge at the trans-Golgi network (TGN), which also functions as an early endosome (EE). Multivesicular bodies (MVBs) are the other main endosomal compartment in plants and serve as prevacuolar compartments (PVCs) or late endosomes (LE) destined for vacuolar degradation (reviewed (1, 5, 6)).Recycling and sorting of plasma membrane proteins is essential for generating the polar localization of auxin efflux transporters (discussed in (7)), formation of the cell plate during cell division (8–11), and in defense such as localized deposition of papilla reviewed in (12, 13). Furthermore, the subcellular localization of transporters and receptors is dynamically regulated. For example, the boron transporter (BOR1) exhibits polar localization and is internalized and degraded under conditions of high boron to reduce toxicity (14, 15). Similarly the receptor-like kinases (RLKs) flagellin-sensing 2 (FLS2) and brassinosteroid insensitive 1 (BRI1), important transmembrane receptors in antibacterial immunity and plant development, respectively, are constitutively endocytosed and recycled to the PM (16–18). Both receptors and transporters are also cargoes of the LE/MVB trafficking route (16) and are probably sorted to the vacuole for degradation (19, 20). Importantly, FLS2 trafficking via the recycling endocytic or the late endocytic route depends on its activation status; inactive receptors are recycled while ligand-activated receptors are sorted to the late endosomal pathway (16). Similarly, the polar sorting of auxin efflux transporters depends on their phosphorylation status (21). These observations illustrate that membrane compartmentalization underpins important aspects of plant cell biology and has initiated a quest toward a better understanding of the endomembrane compartments and the routes and mechanisms by which cargo is trafficked and sorted within the cell.Membrane trafficking within the cell requires complex machinery consisting of a plethora of coat and adaptor proteins, small GTPases, targeting, tethering, and scission factors (reviewed in (22, 23)). Homologues of some animal and yeast and endomembrane regulators have been identified in plants, but the localization and function of many of these remain to be characterized. For example, members of the RAB GTPase family have been shown to have markedly different roles and localizations in plants compared with their animal and yeast homologs (24). Therefore, acquiring localization data for tethering complexes and other regulators in plant systems is essential. In Arabidopsis thaliana, some of these proteins have been developed as useful probes to visualize the different endomembrane compartments by fusion with fluorescent reporters (9, 25–27). These include regulators of trafficking events such as RAB GTPases that are molecular switches responsible for the assembly of tethering and docking complexes and compartment identity. RAB proteins are widely used markers of endomembrane compartments, for example RABD2a/ARA5 labels the Golgi and TGN/EE as well as post-Golgi vesicles (4, 24, 26, 28), RABF2b/ARA7 localizes to TGN/EE and LE (25), RABF1/ARA6 is a marker of the LE/MVB vesicles (25, 29), and RABG3f localizes to MVBs and the tonoplast (26, 30).Fluorescent-tagged marker lines for the live-cell imaging of plant cells have been invaluable in defining the location of proteins within and between organelles and endomembrane compartments (26). However, microscopic investigation of membrane trafficking is limited by throughput, as only few proteins can be studied simultaneously. A powerful approach to large-scale identification of proteins in endomembrane compartments is through subcellular fractionation based on physical properties to directly isolate or enrich for the subcellular compartment of interest. Subcellular fractionation-based proteomics have been successfully used to decipher the steady state and cargo proteomes of, including but not limited to, the ER, the vacuole, PM, mitochondria and chloroplasts, and smaller vesicle-like compartments such as peroxisomes and Golgi (31–41). However, the smaller, transitory vesicles of the secretory and endocytic pathways have proved challenging to purify for reliable proteomic analysis. To overcome this, affinity purification of vesicles was established in animal cells (42, 43) and recently successfully applied in plants in combination with subcellular fractionation. Affinity purification and mass spectrometry (MS) of syntaxin of plants 61 (SYP61)-positive TGN/EE compartments identified 145 proteins specifically enriched in (44), while affinity isolation of VHA-a1-GFP (vacuolar H⁺ ATPase A1) identified 105 proteins associated with the TGN/EE (45). The VHA-A1 affinity purification data were then further refined using density gradient centrifugation to differentiate cargo and steady-state proteins (45).We have further explored affinity purification of fluorescent-tagged markers localizing to defined compartments to identify proteins associated with trafficking. Our motivation was to dissect the trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with small GTPases in the RAB family. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for Golgi/TGN/EE/secretory vesicles, LE/MVB compartments, LE/MVB compartments and LE/MVB/tonoplast, respectively. Additionally, we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles (CCVs) and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast (26, 27, 29, 46, 47) as comparisons. Our objective was to identify transient cargo proteins, tethers, and docking factors associated with dynamic subdomains of the endomembrane system, to supplement better-characterized “steady-state” components, and to identify components of recycling and vacuolar trafficking pathways.