Tommy Nilsson

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum.

The cytosolic coat-protein complex COP-I interacts with cytoplasmic ‘retrieval’ signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.

GTP hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles

Upon addition of GTPγS to in vitro budding reactions, COP I vesicles form but retain their coat, making them easy to isolate and analyze. We have developed an in vitro budding assay that reconstitutes the formation of COP I‐derived vesicles under conditions where GTP hydrolysis can occur. Once formed, vesicles are uncoated and appear functional as they fuse readily with acceptor membranes. Electron microscopy shows a homogeneous population of uncoated vesicles that contain the medial/trans Golgi enzyme α1,2‐mannosidase II. Biochemical quantitation of vesicles reveals that resident Golgi enzymes are up to 10‐fold more concentrated than in donor membranes, but vesicles formed in the presence of GTPγS show an average density of resident Golgi enzymes similar to that seen in donor membranes. We show that the sorting process is mediated by the small GTPase arf‐1 as addition of a dominant, hydrolysis‐deficient arf‐1 Q71L mutant produced results similar to that of GTPγS. Strikingly, the average density of the anterograde cargo protein, polymeric IgA receptor, in COP I‐derived vesicles was similar to that found in starting membranes and was independent of GTP hydrolysis. We conclude that hydrolysis of GTP bound to arf‐1 promotes selective segregation and concentration of Golgi resident enzymes into COP I vesicles.

Identification of rabaptin-5, rabex-5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER

The role of rab33b, a Golgi‐specific rab protein, was investigated. Microinjection of rab33b mutants stabilised in the GTP‐specific state resulted in a marked inhibition of anterograde transport within the Golgi and in the recycling of glycosyltransferases from the Golgi to the ER, respectively. A GST‐rab33b fusion protein stabilised in its GTP form was found to interact by Western blotting or mass spectroscopy with Golgi protein GM130 and rabaptin‐5 and rabex‐5, two rab effector molecules thought to function exclusively in the endocytic pathway. A similar binding was seen to rab1 but not to rab6, both Golgi rabs. In contrast, rab5 was as expected, shown to bind rabaptin‐5 and rabex‐5 as well as the endosomal effector protein EEA1 but not GM130. No binding of EEA1 was seen to any of the Golgi rabs.

Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion

Much recent work has focussed on the role of membrane‐bound components in fusion. We show here that p97 and NSF are sufficient to mediate rapid membrane fusion. Fractionation of cytosol revealed that p97 and its co‐factor, p47, constitutes the major fusion activity. This was confirmed by depleting p97 from the cytosol, which resulted in an 80% decrease in fusion. Using purified protein, p97 or NSF was found to be sufficient to mediate rapid fusion in an ATP‐dependent manner. A regulatory role was observed for their corresponding co‐factors, p47 and α‐SNAP. When present at a molar ratio half of that of the ATPase, both co‐factors increased fusion activity significantly. Intriguingly, at this ratio the ATPase activity of the complex measured in solution was at its lowest, suggesting that the co‐factor stabilizes the ATP state. The fusion event involved mixing of both leaflets of the opposing membranes and contents of liposomes. We conclude from these data that p97, NSF and perhaps other related ATPases catalyse rapid and complete fusion between lipid bilayers on opposing membranes. This highlights a new role for p97 and NSF and prompts a re‐evaluation of current fusion models.

Cisternal maturation and vesicle transport: join the band wagon! (Review)

No cellular organelle has been the subject of as many, as long-lasting or as diverse polemics as the Golgi apparatus. This statement was made by Whaley almost 30 years ago in the book The Golgi Apparatus and still holds true today, perhaps more then ever. Why is this? How come something as mundane as a series of intracellular membrane bound structures continues to fascinate and captivate a large section of the cell biology community? One simple reason (putting polemics aside) is that the secretory pathway appears deceptively simple. Once probed, however, it has a persistent habit of developing into an enigma. Is one then not closer than 30 years ago? In a sense yes, in that one has more components and a better understanding of inherent membrane dynamics, but it is still not known how newly synthesized proteins and lipids make their way from the ER to the plasma membrane. Is it by vesicles, cisternal carriers or transient tubular connections? It has also been learned that newly synthesized proteins are segregated away from the resident components throughout the pathway, but not how. Do coat proteins hold the key? It is understood that the cytoskeleton is important, but not really why. It is known that each Golgi stack is a fully functional unit, but not why stacks are connected laterally into a large ribbon (the Golgi apparatus). This review focuses on how proteins make their way through the pathway, a basic question that remains to be answered.

Protein sorting in the Golgi apparatus: a consequence of maturation and triggered sorting

To explain how resident proteins distribute in peak‐like patterns at various positions in the secretory pathway, Glick and co‐workers postulated that resident proteins comprise different populations (termed kin populations) and that these compete with each other for entering retrograde transport carriers [Glick et al. (1997) FEBS Lett. 414, 177–181]. Using modelling and computer simulation, they could demonstrate that differences in competitiveness sufficed to generate overlapping but distinct peak‐like steady state distributions of the different kin populations across the Golgi stack. In this study, we have tested the robustness of the competition model and find that over‐expression or changes in the number of kin populations affect their overall steady state distributions. To increase the robustness of the system, we have introduced a milieu‐induced trigger for recycling. This allows for a decrease in the coupling between kin populations permitting both over‐expression as well as changes in the number of kin populations. We have also extended the model to include a Golgi to endoplasmic reticulum (ER) recycling pathway and find that only a small amount of resident proteins may recycle at any time without upsetting their observed distributions in the Golgi stack. The biological relevance of a trigger‐induced sorting mechanism and ER recycling is discussed.

16 – Vesicular Transport

Publisher Summary nThis chapter explores the vesicular transport. The newly synthesized proteins destined for the plasma membrane are imported into the ER where they oligomerize after folding, receive N-linked oligosaccharides, and are then checked by the quality control machinery before export from the transitional ER through vesicular intermediates. This chapter puts together a brief note on COPI and COPII, describing a role for coat proteins in cargo selection. The existence of transport vesicles as transport intermediates in the secretory pathway has never been universally accepted. Transport vesicles are supported by a large body of evidence derived from morphological, molecular, genetic as well as biochemical studies. The need for GTP hydrolysis therefore suggests that for proper sorting to take place, coatomer needs to cycle on and off the membrane. This chapter also stresses that the role of cargo molecules in vesicle formation has been so far underscored through their ability to interact directly with coat proteins. Important insights into the mechanism of a complex reaction can also be obtained by studying the kinetics of the process. If this is how sorting occurs remains to be tested but importantly, these studies show that simple parameters can define models that predict the asymmetric distributions, offering clues to how the secretory pathway operates.