Crystal structure of the S.cerevisiae exocyst component Exo70p

The exocyst is an evolutionarily conserved multiprotein complex required for the targeting and docking of post-Golgi vesicles to the plasma membrane. Through its interactions with a variety of proteins, including small GTPases, the exocyst is thought to integrate signals from the cell and signal that vesicles arriving at the plasma membrane are ready for fusion. Here we describe the three-dimensional crystal structure of one of the components of the exocyst, Exo70p, from Saccharomyces cerevisiae at 3.5A resolution. Exo70p binds the small GTPase Rho3p in a GTP-dependent manner with an equilibrium dissociation constant of approximately 70 microM. Exo70p is an extended rod approximately 155 angstroms in length composed principally of alpha helices, and is a novel fold. The structure provides a first view of the Exo70 protein family and provides a framework to study the molecular function of this exocyst component.     

Arabidopsis TRAPPII is functionally linked to Rab-A,but not Rab-D in polar protein trafficking in trans-Golgi network

The trans-Golgi network (TGN) in plant cells is an independent organelle, displaying rapid association and dissociation with Golgi bodies. In plant cells, the TGN is the site where secretory and endocytic membrane trafficking meet. Cell wall components, signaling molecules and auxin transporters have been found to undergo intracellular trafficking around the TGN. However, how different trafficking pathways are regulated and how different cargoes are sorted in the TGN is poorly defined in plant cells. Using a combined approach of genetic and in vivo imaging, we recently demonstrated that Arabidopsis TRAPPII acts in the TGN and is required for polar targeting of PIN2, but not PIN1, auxin efflux carrier in root tip cells. Here, we report that, TRAPPII in Arabidopsis is required for polar distribution of AUX1, an auxin influx carrier in protophloem cells and epidermal cells of Arabidopsis root tips. In yeast cells, TRAPPII serves as a guanine-nucleotide exchange factor (GEF) for Ypt1 and Ypt31/32 in late Golgi trafficking, while in mammalian cells, TRAPPII acts as a GEF for Rab1 (homolog of yeast Ypt1) in early Golgi trafficking. We show here that TRAPPII in Arabidopsis is functionally linked to Rab-A proteins, homologs of yeast Ypt31/32, but not Rab-D proteins, homologs of yeast Ypt1 and animal Rab1 proteins.     

Naegleria’s mitotic spindles are built from unique tubulins and highlight core spindle features

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Are All Multisubunit Tethering Complexes Bona Fide Tethers?

Since the late 1990s, a number of multisubunit tethering complexes (MTCs) have been described that function in membrane trafficking events: TRAPP I, TRAPP II, TRAPP III, COG, HOPS, CORVET, Dsl1, GARP and exocyst. On the basis of structural and sequence similarities, they have been categorized as complexes associated with tethering containing helical rods (CATCHR) (Dsl1, COG, GARP and exocyst) or non‐CATCHR (TRAPP I, II and III, HOPS and CORVET) complexes (Yu IM, Hughson FM. Tethering factors as organizers of intracellular vesicular traffic. Annu Rev Cell Dev Biol 2010;26:137–156). Both acronyms (CATCHR and MTC) imply these complexes tether opposing membranes to facilitate fusion. The main question we will address is: have these complexes been formally demonstrated to function as tethers? If the answer is no, then is it premature or even correct to refer to them as tethers? In this commentary, we will argue that the vast majority of MTCs have not been demonstrated to act as a tether. We propose that a distinction between the terms tether and tethering factor be considered to address this issue.     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

Recycling,clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall‐encapsulated plant cells. We have used super‐resolution and semi‐quantitative live‐cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar‐competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super‐polar recycling. Within the plasma membrane, PINs are recruited into non‐mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin‐dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super‐polar exocytosis have primary importance for PIN polarity maintenance.     

进化细胞生物学的提出及其任务

作者提出应创建一门源于进化生物学与细胞生物学两者的交叉学科一进化细胞生物学(细胞的进化生物学)。其根本任务在于用进化的观点考察真核细胞的一切方面,从它们的起源和演化来认识它们的现在。文中列举了其具体的研究内容,并分析了其研究方法上的特点,指出在这里需要把进化生物学的综合性分析与细胞生物学的实验研究最紧密地结合起来。文中还论述了真核细胞的细胞器的“不进化”现象,指出其根本原因在于进化焦点的转移。     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

Cell biology

A selection of World Wide Web sites relevant to papers published in this issue of Current Opinion in Plant Biology.     

Remodeling the Specificity of an Endosomal CORVET Tether Underlies Formation of Regulated Secretory Vesicles in the Ciliate Tetrahymena thermophila

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The amino acids upstream of NH(2)-terminal dileucine motif play a role in regulating the intracellular sorting of the Class III transporters GLUT8 and GLUT12

Abstract

The transport of glucose across cell membranes is mediated by a family of facilitative glucose transporters (GLUTs). The class III glucose transporters GLUT8 and GLUT12 both contain a similar [DE]XXXL[LI] dileucine sorting signal in their amino terminus. This type of dileucine motif facilitates protein trafficking to various organelles or to the plasma membrane via interactions with adaptor protein (AP) complexes. The [DE]XXXL[LI] motif in GLUT8 is thought to direct it to late endosomal/lysosomal compartments via its interactions with AP1 and AP2. Unlike GLUT8, the [DE]XXXL[LI] motif does not direct GLUT12 to a lysosomal compartment. Rather, GLUT12 resides in the Golgi network and at the plasma membrane. In a previous study, we found that exchanging the XXX (TQP) residues in GLUT8 with the corresponding residues in GLUT12 (GPN) resulted in a dramatic missorting of GLUT8 to the cell surface. We postulated that the XXX amino acids upstream of the dileucine motif in GLUT8 influence the degree of interaction between the [DE]XXXL[LI] motif and adaptor proteins. To further explore its trafficking mechanisms, we created mutant constructs to identify the role that each of the individual XXX amino acids has for regulating the intracellular sorting of GLUT8. Here we find that the XXX amino acids, specifically the position of a proline -2 from the dileucine residues, influence the affinity of APs for GLUT8 and GLUT12.     

Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins

Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.