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Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins Via Tubulovesicular Recycling Endosomes Rytis Prekeris,* Judith Klumperman,‡ Yu A

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Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins Via Tubulovesicular Recycling Endosomes Rytis Prekeris,* Judith Klumperman,‡ Yu A Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins via Tubulovesicular Recycling Endosomes Rytis Prekeris,* Judith Klumperman,‡ Yu A. Chen,* and Richard H. Scheller* *Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5428; and ‡Medical School, University of Utrecht, Institute for Biomembranes, 3584CX Utrecht, The Netherlands Abstract. Endocytosis-mediated recycling of plasma oles, where it is often found in clathrin-coated mem- membrane is a critical vesicle trafficking step important brane areas. Furthermore, anti-syntaxin 13 antibody in- in diverse biological processes. The membrane traffick- hibits transferrin receptor recycling in permeabilized ing decisions and sorting events take place in a series of PC12 cells. Immunoprecipitation of syntaxin 13 re- heterogeneous and highly dynamic organelles, the en- vealed that, in Triton X-100 extracts, syntaxin 13 is dosomes. Syntaxin 13, a recently discovered member of present in a complex(es) comprised of bSNAP, VAMP the syntaxin family, has been suggested to play a role in 2/3, and SNAP-25. This complex(es) binds exogenously mediating endosomal trafficking. To better understand added aSNAP and NSF and dissociates in the presence the function of syntaxin 13 we examined its intracellu- of ATP, but not ATPgS. These results support a role lar distribution in nonpolarized cells. By confocal im- for syntaxin 13 in membrane fusion events during the munofluorescence and electron microscopy, syntaxin recycling of plasma membrane proteins. 13 is primarily found in tubular early and recycling en- dosomes, where it colocalizes with transferrin receptor. Key words: vesicular transport • endosomes • protein Additional labeling is also present in endosomal vacu- recycling • membrane trafficking • syntaxin iological membranes are used to establish func- dermal growth factor (38, 39, 59) become highly concen- tional compartments in eucaryotic organisms. The trated in clathrin-coated vesicles. After endocytosis, inter- B plasma membrane serves as an efficient and spe- nalized proteins, lipids, and solutes are recycled back to cific filter separating the cytoplasm from the extracellular the cell surface or routed to the degradative pathway. The space. As a part of mechanisms used to maintain homeo- membrane trafficking decisions and sorting events take stasis, cells constantly internalize plasma membrane which place in a series of heterogeneous and highly dynamic or- often includes molecules from the outside world. The best ganelles, the endosomes (38, 47). Separation of recycling characterized mechanism of entry into the cell, endocyto- proteins from proteins destined for lysosomal degradation sis, occurs mainly through the formation of clathrin-coated takes place, at least in part, within early endosomes (EE), vesicles (43, 44). A key event in this process is recruitment also known as sorting endosomes, located in the periphery of cytosolic clathrin to the membrane, where it associates of the cell (26, 37). The EE is a compartment comprised with protein complexes called adaptor proteins (12, 44). of two general domains, a vacuolar domain adjacent to a Many of the membrane receptors for extracellular ligands, network of tubules and vesicles which can be dispersed such as transferrin (Tf),1 low-density lipoprotein, and epi- throughout the cytoplasm (38). Recycling proteins enter these tubular extensions by a process which appears to in- Address correspondence to R.H. Scheller, Howard Hughes Medical Insti- volve clathrin coat-dependent budding. The vacuolar do- tute, Department of Molecular and Cellular Physiology, Stanford Univer- main, on the other hand, contains proteins destined for sity School of Medicine, Stanford, CA 94305-5428. Tel.: (650) 723-9075. degradation, and either directly matures into the late en- Fax: (650) 725-4436. E-mail: [email protected] dosomes (LE) and lysosomes, or shuttles proteins to these 1. Abbreviations used in this paper: BFA, brefeldin A; EE, early endo- organelles through endosomal carrier vesicles. From the somes; LE, late endosomes; NEM, N-ethylmaleimide; NRK, normal rat EE, recycling molecules can be rapidly shuttled back to kidney; NSF, N-ethylmaleimide–sensitive factor; PNS, postnuclear super- the plasma membrane or continue to the separate tubu- natant; RE, recycling endosomes; SLO, streptolysin-O, SNAP, soluble lovesicular compartment located in the pericentriolar re- NSF attachment protein; SNAP-25, synaptosomal-associated protein of 25 kD; SNARE, soluble NSF attachment protein receptor; t-SNARE, target gion of cell, known as recycling endosomes (RE) (21) or SNARE; Tf, transferrin; TfR, transferrin receptor; VAMP, vesicle-associ- peri-Golgi recycling compartment (27, 29). The transit ated membrane protein. through RE proceeds with slower kinetics than transport The Rockefeller University Press, 0021-9525/98/11/957/15 $2.00 The Journal of Cell Biology, Volume 143, Number 4, November 16, 1998 957–971 http://www.jcb.org 957 through EE (14, 49) and depends on an intact microtubu- vacuolar compartment, and vesicles delivering alkaline lar network (20, 27, 29, 67). The definition of the bound- phosphatase to the vacuoles (15). In addition, the yeast aries between the highly dynamic EE and RE remains v-SNARE Vti1p interacts with several t-SNAREs, includ- controversial, and at least in some cell types all endosomes ing Pep12p (Golgi to endosomes), Sed5p (ER to Golgi), have been proposed to form a continuous tubulovesicular and Tgi1p/Tgi2p (trans-Golgi). These data suggest that in- network (28). Indeed, the RE retain the highly tubular na- dividual SNAREs operate in multiple trafficking steps and ture observed in parts of the EE. Thus, it remains to be de- in multiple complexes with other proteins. Thus, an under- termined whether EE and RE comprise truly distinct or- standing of the proteins that regulate vesicular trafficking ganelles. On the other hand, the existence of proteins relies not only on the identification of t- and v-SNARES, which are restricted to specific classes of endosomes ar- but also on the characterization of the combinatorial inter- gues in favor of the existence of a biochemically and func- actions between them. tionally distinct set of compartments. For instance, the Despite the progress in our understanding of ER to small GTPase rab4 (14) and rab5 (11) are associated only Golgi vesicular trafficking and exocytosis, very little is with the EE, whereas rab11 (60) is thought to be specific known about molecular events involved in regulating the for the RE. Moreover, although the EE contain proteins docking and fusion in the endocytic pathway of mamma- destined for recycling as well as degradation, the RE con- lian cells. Only one SNARE, VAMP3, has been shown to tain proteins largely destined for transport back to the cell have an endosomal localization (34). However, it remains surface (67). to be determined which endosomal syntaxins interact with Understanding the intricate and dynamic organization VAMP3. So far three putative endosomal t-SNAREs have of endosomal compartments is dependent on our ability to been characterized: syntaxin 7 (63, 66), syntaxin 12 (57), establish relationships between the morphological descrip- and syntaxin 13 (1). Syntaxin 12 and syntaxin 13 share tions and biochemical definitions of the different traffick- 98% homology and are likely to be orthologues. All three ing steps through the endocytic pathway. In recent years a syntaxins were cloned using sequences obtained from a set of proteins has emerged whose role is to mediate and human expressed sequence tag database and are suggested regulate intracellular membrane fusion events. Vesicle- to play a role in endosomal trafficking based on their ho- associated membrane proteins (VAMPs) and syntaxins, mology to Pep12p, the yeast t-SNARE involved in Golgi also known as soluble N-ethylmaleimide–sensitive factor to endosome trafficking (5). However, only syntaxin 7 has (NSF) attachment protein (SNAP) receptors or SNAREs, been linked to the endocytic pathway, through its localiza- have been suggested to be important in determining the tion on EE (66). Moreover, the other regulatory proteins specificity of vesicle transport and to mediate membrane which interact with syntaxins 7 and 13 remain to be deter- fusion (6, 54, 55). Soluble NSF attachment protein recep- mined. tor (SNARE) proteins were initially divided into two func- Here we characterize the subcellular distribution and tional classes, target (t)-SNAREs of the syntaxin family function of syntaxin 13, a putative endosomal SNARE. By and vesicle (v)-SNAREs of the VAMP family, based on immunofluorescence and electron microscopy we local- their putative role as vesicular or target membrane pro- ized syntaxin 13 to tubular extensions of early endosomes, teins, respectively. The membrane fusion event, at least in as well as recycling endosomes. Moreover, using a Tf recy- nerve terminals, has been proposed to be mediated by cling assay in permeabilized PC12 cells, we demonstrate formation of a very stable core complex, comprised of that syntaxin 13 is directly involved in the recycling of VAMP2, syntaxin 1, and synaptosomal-associated protein plasma membrane proteins. Finally, using immunoprecipi- of 25 kD (SNAP-25), which brings the membranes to- tation and microsequencing techniques, we identify puta- gether in direct opposition (22, 33). It is believed that the tive syntaxin 13–interacting proteins.
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