Apoplastic exosome-like vesicles: A new way of protein secretion in plants?

The presence of apoplastic proteins without predicted signal peptide in the gene sequence suggests the existence of protein secretion independent of the ER/Golgi classical route. In animals, one of the pathways proposed for alternative protein secretion involves the release of exosomes to the extracellular space. Although this pathway has not been dissected in plants some indirect evidence is emerging. We have reported that apoplastic fractions of sunflower seeds contain exosome-like vesicles. Besides, these vesicles are enriched in the lectin Helja, which is immunolocalized in the extracellular space even if it the protein has no predicted signal peptide. Here we show that Helja is not glycosylated and its secretion is insensitive to brefeldin A, two of the major characteristics to discard ER/Golgi-mediated protein transport. Moreover, the levels of Helja in sunflower extracellular vesicles are not affected by brefeldin A treatment. Our results suggest that Helja could be exported through an exosome-mediated pathway and point out that this mechanism may be responsible for the secretion of at least part of the leaderless proteins detected in the extracellular compartment of plants.     

Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca(2+)-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca(2+), a approximately 35-40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca(2+), and the interaction is direct and optimal at 1 microM Ca(2+). Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca(2+)-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.     

非经典的蛋白质分泌途径

蛋白质分泌是细胞间进行信息传递的重要方式之一。一般的分泌蛋白在其N端都具有信号肽序列,可以通过经典的内质网-高尔基体途径进行运输,并最终分泌到细胞外。近来发现细胞内存在另一类分泌蛋白,它们能够分泌到细胞外发挥功能,但却没有典型的信号肽序列,被称为非经典分泌蛋白。越来越多的证据表明,这类蛋白质的分泌有其独特的机制。本综述了这类蛋白质的各种分泌机制及可能的生理意义。     

Regulation of protein trafficking by glycosylphosphatidylinositol valence in African trypanosomes

The structure, biosynthesis, and attachment of glycosylphosphatidylinositol (GPI) anchors were all first determined for the variant surface glycoprotein (VSG) of African trypanosomes, and all of the basic aspects of this work have been shown to be universal in eukaryotic organisms. However, the role of GPI anchors in protein trafficking within trypanosomes has lagged behind the more standard eukaryotic model systems such as yeast and polarized epithelial cells. Trypanosomes are also highly polarized cells in which all endocytosis and exocytosis intersect at a discrete domain of the plasma membrane, the flagellar pocket. Within these convergent pathways trafficking of GPI anchored proteins correlates strongly with valence: homodimeric VSG with two GPIs is stably incorporated into the cell surface coat, heterodimeric transferrin receptor with a single GPI is found in the flagellar pocket and is slowly delivered to the lysosome for degradation, and recombinant GPI minus VSG reporters are rapidly degraded in the lysosome. Here we summarize recent data confirming this correlation using a tool kit of recombinant GPI anchored reporters, including a reporter designed to be conditionally modulated between a GPI valence of one and two.     

菌丝形态发育：丝状真菌细胞工厂遗传改造的新切入点

丝状真菌是一类广泛存在于自然界中的真核微生物,一些丝状真菌因具有强大的蛋白分泌能力或者可以有效合成许多活性物质被开发为“细胞工厂”,常被用于酶制剂、重组蛋白、有机酸以及次级代谢产物的生产。丝状真菌菌体的生长形态对工业发酵具有关键意义,对发酵产品的品质及发酵过程的控制均有显著影响。本课题组前期研究发现丝状真菌的菌丝形态直接影响着菌体的蛋白分泌,菌丝分枝的增多会使液体发酵时蛋白质的分泌量增加。随着丝状真菌形态研究的深入,对改变真菌菌丝体形态以提高发酵过程中目标代谢物产量的研究日益增多。尽管在真菌发酵形态与生产力的关系方向已有少量综述,但该领域的研究发展非常迅速,亟待更新。本文在对国内外相关研究报告进行大量调研的基础上,结合作者科研团队自己的研究发现,系统综述了丝状真菌的形态特征、真菌形态对工业发酵的影响及菌丝形态调控的方法和策略,以期增强国内相关学者对丝状真菌菌丝形态发育的认识,为合理开发适用于工业发酵的工程菌株提供思路。     

Protein Trafficking to Apical Organelles of Malaria Parasites – Building an Invasion Machine

Malaria is caused by four species of apicomplexan protozoa belonging to the genus Plasmodium. These parasites possess a specialized collection of secretory organelles called rhoptries, micronemes and dense granules (DGs) that in part facilitate invasion of host cells. The mechanism by which the parasite traffics proteins to these organelles as well as regulates their secretion has important implications for understanding the invasion process and may lead to development of novel intervention strategies. In this review, we focus on emerging data about trafficking signals, mechanisms of biogenesis and secretion. At least some of these are conserved in higher eukaryotes, suggesting that rhoptries, micronemes and DGs are related to organelles such as secretory lysosomes that are well known to mainstream cell biologists.     

Regulation of developmental intercellular signalling by intracellular trafficking

Universal trafficking components within the cell can be recruited to coordinate and regulate the developmental signalling cascades. We will present ways in which the intracellular trafficking machinery is used to affect and modulate the outcome of signal transduction in developmental contexts, thus regulating multicellular development. Each of the signalling components must reach its proper intracellular destination, in a form that is properly folded and modified. In many instances, the ability to bring components together or segregate them into distinct compartments within the cell actually provides the switch mechanism to turn developmental signalling pathways on or off. The review will begin with a focus on the signal-sending cells, and the ways in which ligand trafficking can impinge on the signalling outcome, via processing, endocytosis and recycling. We will then turn to the signal-receiving cell, and discuss mechanisms by which endocytosis can affect the spatial features of the signal, and the compartmentalization of components downstream to the receptor.