Genetic variation is the major mechanism behind adaptation and evolutionary change. As most proteins operate through interactions with other proteins, changes in protein complex composition and subunit sequence provide potentially new functions. Comparative genomics can reveal expansions, losses and sequence divergence within protein-coding genes, but in silico analysis cannot detect subunit substitutions or replacements of entire protein complexes. Insights into these fundamental evolutionary processes require broad and extensive comparative analyses, from both in silico and experimental evidence. Here, we combine data from both approaches and consider the gamut of possible protein complex compositional changes that arise during evolution, citing examples of complete conservation to partial and total replacement by functional analogues. We focus in part on complexes in trypanosomes as they represent one of the better studied non-animal/non-fungal lineages, but extend insights across the eukaryotes by extensive comparative genomic analysis. We argue that gene loss plays an important role in diversification of protein complexes and hence enhancement of eukaryotic diversity.

• Keywords
• constructive neutral evolution, evolutionary divergence, evolutionary mechanisms, gene replacement, molecular evolution, protein complexes,
• MeSH
• Eukaryota * genetics   MeSH
• Phylogeny MeSH
• Genomics MeSH
• Evolution, Molecular * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Vesicle exocytosis underpins signaling and development in plants and is vital for cell expansion. Vesicle tethering and fusion are thought to occur sequentially, with tethering mediated by the exocyst and fusion driven by assembly of soluble NSF attachment protein receptor (SNARE) proteins from the vesicle membrane (R-SNAREs or vesicle-associated membrane proteins [VAMPs]) and the target membrane (Q-SNAREs). Interactions between exocyst and SNARE protein complexes are known, but their functional consequences remain largely unexplored. We now identify a hierarchy of interactions leading to secretion in Arabidopsis (Arabidopsis thaliana). Mating-based split-ubiquitin screens and in vivo Förster resonance energy transfer analyses showed that exocyst EXO70 subunits bind preferentially to cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distribution and suppressed vesicle traffic similarly to the dominant-negative truncated protein SYP121ΔC, which blocks secretion at the plasma membrane. These phenotypes are consistent with the epistasis of exo70A1 in the exo70A1 syp121 double mutant, which shows decreased growth similar to exo70A1 single mutants. However, the exo70A1 vamp721 mutant showed a strong, synergy, suppressing growth and cell expansion beyond the phenotypic sum of the two single mutants. These data are best explained by a hierarchy of SNARE recruitment to the exocyst at the plasma membrane, dominated by the R-SNARE and plausibly with the VAMP721 longin domain as a nexus for binding.

• MeSH
• Arabidopsis cytology   genetics   growth & development   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Exocytosis physiology   MeSH
• Plants, Genetically Modified MeSH
• Mutation MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Qa-SNARE Proteins genetics   metabolism   MeSH
• R-SNARE Proteins genetics   metabolism   MeSH
• SNARE Proteins genetics   metabolism   MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• EXO70A1 protein, Arabidopsis MeSH Browser
• PEN1 protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• Qa-SNARE Proteins MeSH
• R-SNARE Proteins MeSH
• SNARE Proteins MeSH
• VAMP721 protein, Arabidopsis MeSH Browser

Background: The eukaryotic endomembrane system most likely arose via paralogous expansions of genes encoding proteins that specify organelle identity, coat complexes and govern fusion specificity. While the majority of these gene families were established by the time of the last eukaryotic common ancestor (LECA), subsequent evolutionary events has moulded these systems, likely reflecting adaptations retained for increased fitness. As well as sequence evolution, these adaptations include loss of otherwise canonical components, the emergence of lineage-specific proteins and paralog expansion. The exocyst complex is involved in late exocytosis and additional trafficking pathways and a member of the complexes associated with tethering containing helical rods (CATCHR) tethering complex family. CATCHR includes the conserved oligomeric Golgi (COG) complex, homotypic fusion and vacuole protein sorting (HOPS)/class C core vacuole/endosome tethering (CORVET) complexes and several others. The exocyst is integrated into a complex GTPase signalling network in animals, fungi and other lineages. Prompted by discovery of Exo99, a non-canonical subunit in the excavate protist Trypanosoma brucei, and availability of significantly increased genome sequence data, we re-examined evolution of the exocyst. Methods: We examined the evolution of exocyst components by comparative genomics, phylogenetics and structure prediction. Results: The exocyst composition is highly conserved, but with substantial losses of subunits in the Apicomplexa and expansions in Streptophyta plants, Metazoa and land plants, where for the latter, massive paralog expansion of Exo70 represents an extreme and unique example. Significantly, few taxa retain a partial complex, suggesting that, in general, all subunits are probably required for functionality. Further, the ninth exocyst subunit, Exo99, is specific to the Euglenozoa with a distinct architecture compared to the other subunits and which possibly represents a coat system. Conclusions: These data reveal a remarkable degree of evolutionary flexibility within the exocyst complex, suggesting significant diversity in exocytosis mechanisms.

• Keywords
• Exocytosis, comparative genomics, eukaryotes, exocyst, membrane transport, molecular evolution,
• Publication type
• Journal Article MeSH

The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell.

• MeSH
• Cell Membrane metabolism   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Exocytosis genetics   MeSH
• Phylogeny MeSH
• Spectrometry, Mass, Electrospray Ionization MeSH
• Microscopy, Confocal MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Proteomics methods   MeSH
• Pollen Tube genetics   growth & development   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Proteins classification   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Nicotiana genetics   metabolism   MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Gene Expression Regulation, Developmental MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Isoforms MeSH
• Plant Proteins MeSH