Comments on: Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport

Vicky Miller (Postdoc in the Stephens lab) and I (David) have written the following comments on this paper that also appear on the Faculty of 1000 site. Vicky is also on Twitter @Dr_VickyMiller

Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport.

Cutrona MB, Beznoussenko GV, Fusella A, Martella O, Moral P, Mironov AA.

Traffic 2013; DOI: 10.1111/tra.12060 PMID: 23433038

This paper describes studies of trafficking of secretory cargo from the ER in cells depleted of the small GTPase Sar1, an essential component of the COPII coat. A COPII-independent mechanism of secretory trafficking would mark a step-change in our understanding of secretory protein trafficking in cells. While we do not think that this paper requires such a re-evaluation of our thinking, it does provide some thought provoking data. By disrupting the normal mechanism of transport the authors seek to reveal COPII-independent mechanisms and challenge the primacy of COPII-dependent transport. The most important point to bear in mind when reading this paper is that siRNA knock-downs are unable to generate a complete depletion of any protein and that residual Sar1 may influence some of the results seen. Indeed in our opinion, one cannot conclusively draw the conclusion that COPII-independent ER export pathways exist in cells from this work alone.

The authors confirm that Sar1-depletion effectively inhibits COPII vesicle formation (demonstrated by EM and reduced immunofluorescence staining of COPII components). They see changes to both ER-Golgi intermediate compartment (ERGIC) formation (the compartment between the Golgi and the ER) and Golgi organisation (resulting in formation of mini-stacks), also in agreement with a reduction in secretory trafficking. Despite this, pulse-chase experiments show no reduction in the total amount of protein secretion in Sar1-depleted cells. From this, the authors conclude that alternative transport mechanisms are being used. The virus glycoprotein VSV-G also passes from the ER to the plasma membrane in Sar1-depleted cells, even when additionalCOPII subunits (Sec23A and B) are depleted in addition to Sar1A and B. Based on these and other experiments, the authors propose a COPI-dependent replacement pathway that takes VSV-G to the Golgi. Export of procollagen transport is inhibited by Sar1-depletion, but as collagens require specialized COPII-coated vesicles to accommodate their large size, they therefore are not representative of typical COPII trafficking (Jin et al., 2012). Furthermore, many other studies support the notion that procollagen secretion is exquisitely sensitive to perturbation of the early secretory pathway (Smits et al., 2010; Townley et al., 2008; Venditti et al., 2012). 

These data do support the concept of a “short-loop” ER-to-Golgi trafficking pathway that involves juxtanuclear ER and possible even ER-Golgi contact sites. It is as yet unclear how possible mechanisms such as kiss and run could retainsufficient selectivity to prevent non-selective transport. It remains possible however that the small remaining amount of COPII proteins in the RNAi experiments described here is sufficient to direct COPII-dependent selectivity.

In summary the paper presents some interesting findings. Complete removal of Sar1 from cells by gene deletion is required to examine fully the existence ofCOPII-independent trafficking pathways and the relative importance of COPII-trafficking in cells. 

References

Jin, L., K.B. Pahuja, K.E. Wickliffe, A. Gorur, C. Baumgärtel, R. Schekman, and M. Rape. 2012. Ubiquitin-dependent regulation of COPII coat size and function. Nature 482(7386):495-500. doi: 10.1038/nature10822

Smits, P., A.D. Bolton, V. Funari, M. Hong, E.D. Boyden, L. Lu, D.K. Manning, N.D. Dwyer, J.L. Moran, M. Prysak, B. Merriman, S.F. Nelson, L. Bonafe, A. Superti-Furga, S. Ikegawa, D. Krakow, D.H. Cohn, T. Kirchhausen, M.L. Warman, and D.R. Beier. 2010. Lethal skeletal dysplasia in mice and humans lacking the golginGMAP-210. N. Engl. J. Med. 362:206-216.

Townley, A.K., Y. Feng, K. Schmidt, D.A. Carter, R. Porter, P. Verkade, and D.J. Stephens. 2008. Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development. J. Cell Sci. 121:3025-3034.

Venditti, R., T. Scanu, M. Santoro, G. Di Tullio, A. Spaar, R. Gaibisso, G.V. Beznoussenko, A.A. Mironov, A. Mironov, Jr., L. Zelante, M.R. Piemontese, A. Notarangelo, V. Malhotra, B.M. Vertel, C. Wilson, and M.A. De Matteis. 2012. Sedlin controls the ER export of procollagen by regulating the Sar1 cycle. Science. 337:1668-1672.

       

Full disclosure: David is a member of the “Traffic” editorial board but had not role in the editing or reviewing of this paper.

These comments are also published on F1000 Prime

Evaluation: A pseudoatomic model of the COPII cage

I evaluated this paper for F1000 Prime:

Noble AJ, Zhang Q, O’Donnell J, Hariri H, Bhattacharya N, Marshall AG, Stagg SM. (2013) A pseudoatomic model of the COPII cage obtained from cryo-electron microscopy and mass spectrometry. Nature Structural and Molecular Biology 20(2):167-73

This paper describes a technical tour de force that elucidates some of the finer detail of the molecular structure of the assembled COPII coat. The Stagg lab have obtained a 12-Å structure of the human COPII cage from cryo-electron microscopy and layered on top of this data from hydrogen deuterium exchange (HDX) experiments to define the flexible regions of the assembled structure. The structure was made possible in part by a neat gradient fixation protocol to isolate assembled cages from aggregated material (GraFix, described in (Kastner et al., 2008)). Molecular dynamics flexible fitting of the previous crystallographic structure of the Sec13-31 complex to the EM data provided clear insight into the formation of the vertex elements of the assembled coat. Specifically, the authors demonstrate that Sec13-Sec31 unit has an intrinsic “polarity” within the assembled coat with one end tightly packed and the pother more loosely integrated. This resulted in the identification of a further contact site at the vertex region that reveals a less significant role for Sec13 and a greater contact area through Sec31 than has been previously suggested (Stagg et al., 2008). This has the potential to explain data that suggest that the requirement for Sec13 in vivo is not as stringent as one might expect ((Copic et al., 2012; Townley et al., 2008)). The loose packing evident within the edge element of the Sec31 alpha-solenoid could flex to accommodate unusually large cargo. Overall, this paper is impressive from a technical perspective as well as for the insight it provides into COPII assembly.

Copic, A., C.F. Latham, M.A. Horlbeck, J.G. D’Arcangelo, and E.A. Miller. 2012. ER cargo properties specify a requirement for COPII coat rigidity mediated by Sec13p. Science. 335:1359-1362.

Kastner, B., N. Fischer, M.M. Golas, B. Sander, P. Dube, D. Boehringer, K. Hartmuth, J. Deckert, F. Hauer, E. Wolf, H. Uchtenhagen, H. Urlaub, F. Herzog, J.M. Peters, D. Poerschke, R. Luhrmann, and H. Stark. 2008. GraFix: sample preparation for single-particle electron cryomicroscopy. Nature methods. 5:53-55.

Stagg, S.M., P. LaPointe, A. Razvi, C. Gurkan, C.S. Potter, B. Carragher, and W.E. Balch. 2008. Structural basis for cargo regulation of COPII coat assembly. Cell. 134:474-484.

Townley, A.K., Y. Feng, K. Schmidt, D.A. Carter, R. Porter, P. Verkade, and D.J. Stephens. 2008. Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development. J. Cell Sci. 121:3025-3034.

Evaluated: The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation.

The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation.
McMahon C, Studer SM, Clendinen C, Dann GP, Jeffrey PD, Hughson FM. (2012)
J Biol Chem. in press. PMID: 23109340 DOI: 10.1074/jbc.M112.420141

This article caught my eye for two reasons: first, it maps at high resolution the likely interface between the guanine nucleotide exchange factor Sec12 and the small GTP binding protein Sar1. This interaction is critical for Sar1 activation, the essential precursor to COPII vesicle formation at the endoplasmic reticulum. Second, and where this paper is really worth a look is in the finding that this “K loop” on Sec12 that contacts Sar1 also binds a potassium ion. Furthermore, this K+ ion is essential for the catalytic activity of Sec12 towards Sar1. This paper provides a great example of the value of crystallography on proteins for which there is a very strongly predicted structure.

A Golgi anchor for dynein

I started my lab in 2001 with the aim of defining the mechanisms that link endomembranes, particularly those operating in membrane trafficking between the ER and Golgi, to the microtubule cytoskeleton. This month in Developmental Cell, Adam Linstedt’s lab published the following paper:

Golgin160 Recruits the Dynein Motor to Position the Golgi Apparatus.

Smita Yadav, Manojkumar A. Puthenveedu, Adam D. Linstedt

Developmental Cell

Volume 23, Issue 1, 17 July 2012, Pages 153–165 
http://dx.doi.org/10.1016/j.devcel.2012.05.023
,

I must say that I am very impressed with this paper – it is precisely the one that I wish we had published from my lab. Of course there is much more to do but this is a definitive answer to a question. Nicely done.

Yadav and colleagues set out by defining a role for golgin160 (also known as golgin-160 in the literature) in minus end directed movement of Golgi membranes using siRNA depletion. They also showed that dynein is no longer recruited to Golgi membranes in the absence of golgin160 using an antibody against the dynein light chain Tctex-1. This is no mean feat and immuno-detection of dynein is notoriously difficult and unreliable. They also showed that golgin160 interacts through its own C-terminal coiled coil domain with the dynein intermediate chain subunit. This interaction is direct with a relatively weak (~1 micromolar) affinity.

Golgin160 is a peripheral membrane protein which of course leaves open the question of how it is recruited to membrane to subsequently drive recruitment of dynein. The answer turned out to be Arf1, a small GTP binding protein that operates in ER-to-Golgi transport, primarily to recruit the COPI complex. COPI drives retrograde trafficking from the Golgi to the ER. The N-terminal domain of Golgin160 (i.e. not that which binds dynein) binds preferentially to active GTP-bound Arf1. Consistent with this hierarchical mechanism, golgin160 is not required for the recruitment of GBF1, the main Arf1-activator at this point in the cell¹.

Finally the authors show that Golgin160 dissociates from Golgi membranes during mitosis. This is perhaps not surprising given that it is well known that COPI-dependent membrane traffic is largely arrested during mitosis^{2, 3}. However, it does provide a nice mechanism to drive Golgi disassembly during mitosis. Inhibition (or at least down-regulation) of Arf1 activation would lead to a loss of golgin160 from membranes which would in turn lead to a failure to recruit dynein. This would mean reduced minus end motility and consequently a failure to maintain the Golgi as a juxtanuclear organelle.

These data are very clear and convincing. There are also some nice nuances for aficionados. Importantly the fact that this process relies on Arf1 activation at the start suggests that golgin160 might indeed be a more universal membrane anchor for dynein operating at the Golgi itself as well as in ER-to-Golgi trafficking. My own lab showed previously that the COPII coat⁴ can recruit dynactin, a key accessory factor to dynein that is also required for ER-to-Golgi transport⁵. However, the COPII complex is only involved very locally at ER exit sites⁶ and so another factor must be involved as early as the ER-Golgi intermediate compartment (ERGIC). This factor looks likely to be golgin160. In agreement with this, Yadav et al find that dynactin can be identified in complex with golgin160 and dynein. Whether there is an additional membrane anchor for dynactin, or whether this role is performed by spectrin for example⁷, remains to be defined. Golgin160 is also cleaved during apoptosis by caspase-2⁸ so these new data suggest an obvious mechanism by which this would cause rapid dissociation of the Golgi.

Other questions do remain. How does this golgin160 pathway relate to what we know about the role of Cdc42 and the COPI coat in the recruitment of dynein^{9, 10}.

References:

1.             Szul T, et al. Traffic (2005);6:374-385.

2.             Altan-Bonnet N, et al. Mol Biol Cell (2006);17:990-1005.

3.             Altan-Bonnet N, et al. Proc Natl Acad Sci U S A (2003);100:13314-13319.

4.             Jensen D, Schekman R. J Cell Sci (2011);124:1-4.

5.             Watson P, et al. Nat Cell Biol (2005);7:48-55.

6.             Budnik A, Stephens DJ. FEBS Lett (2009);583:3796-3803.

7.             Holleran EA, et al. J Cell Biol (1996);135:1815-1829.

8.             Mancini M, et al. The Journal of cell biology (2000);149:603-612.

9.             Hehnly H, et al. Traffic (2010);11:1067-1078.

10.          Chen JL, et al. J Cell Biol (2005);169:383-389.

F1000 evaluation: ER Cargo Properties Specify a Requirement for COPII Coat Rigidity Mediated by Sec13p

I have just evaluated the following paper from Faculty of 1000:

ER Cargo Properties Specify a Requirement for COPII Coat Rigidity Mediated by Sec13p.

Alenka Čopič, Catherine F. Latham, Max A. Horlbeck, Jennifer G. D’Arcangelo, Elizabeth A. Miller
Science 2012
PMID 22300850
DOI 10.1126/science.1215909

This fascinating paper defines a link between structural features of an assembling vesicle coat complex with the cargo to be incorporated. Sec13 is an essential component of the COPII coat that drives export of secretory cargo from the endoplasmic reticulum (ER). The requirement for this gene can be bypassed by a series of suppressor mutations in mutants known as Bypass-Sec13 (Bst) (first identified by Chris Kaiser’s lab {1}). The authors took a genetic approach in yeast (using synthetic gene arrays) to identify all of the genes that alleviate the defects caused by loss of Sec13. As in the previous work from Kaiser et al., Miller and colleagues found that all identified genes relate to the synthesis and export of glycosylphosphatidylinositol (GPI)-anchored proteins from the ER. This provides a direct link between the presence of cargo in the inner leaflet (where GPI-anchored proteins reside) and the role of the coat complex on the cytosolic face of the membrane in the formation of vesicles. It was hypothesized that the absence of these cargo proteins from the sites of vesicle formation leads to more readily deformable membranes, eliminating the need for Sec13, the conclusion here being that Sec13 is required to enhance rigidity of the COPII coat to drive membrane deformation under normal conditions. Using a series of elegant mutations in Sec13 and its binding partner Sec31, the authors show quite convincingly that this model of structural rigidity holds true. The finding that Sec13 is required in this way also provides some insight into the apparent selective requirement for Sec13 in the secretion of procollagen (see ref {2}, on which I appear as an author). The structural rigidity of the procollagen triple helix might dictate the requirement for similar structural rigidity within the assembling COPII coat.

References:

{1} Elrod-Erickson and Kaiser, Mol Biol Cell 1996, 7:1043-58 [PMID:8862519].

{2} Townley et al. J Cell Sci 2008, 121:3025-34 [PMID:18713835].

 

The original evaluation can be found here.

F1000 Evaluation (yes, another one)

This time, I have evaluated the following paper. Also on the F1000 site at: 
http://f1000.com/13688961

Kim SD, Pahuja KB, Ravazzola M, Yoon J, Boyadjiev SA, Hammamoto S, Schekman R, Orci L, Kim J.
The SEC23-SEC31 interface plays a critical role for export of procollagen from the endoplasmic reticulum.
J Biol Chem 2012
PMID 22298774 DOI 10.1074/jbc.M111.283382

This paper shows very nicely that the rate of Sar1 GTPase activity within the coat protein complex II (COPII) coat is central to the mechanism of packaging the large cargo procollagen at the endoplasmic reticulum (ER). It is unclear how the canonical COPII system is capable of packaging large cargoes such as procollagen (a 300nm rod-like structure) when it seems geared towards the production of 60-90nm vesicles. Here, Kim and colleagues exploit a mutation in the Sec23A component of the COPII coat that causes cranio-lenticulo-sutural dysplasia (a disease in which there is a pronounced failure to deposit collagenous extracellular matrix). During COPII assembly the inner (Sec23-Sec24) and outer (Sec13-Sec31) layers of the COPII coat both impact on Sar1 GTPase activity. The authors show that, compared to the wild-type protein, the Sec23A-M702V mutation causes an acceleration of Sar1 GTPase activity. This mutation lies at the contact site between Sec23 and Sec31, suggesting a role in coupling inner and outer layers of the coat.

These data support a model where slower assembly or greater stability of the COPII coat is required to package procollagen. The work also supports the notion that procollagen becomes encapsulated within a COPII-coated carrier, but visualization of such a carrier remains elusive.

This work builds very nicely on previous work in this area which was summarized in this recent review {1}.

References:

{1} Zanetti et al. Nat Cell Biol 2012, 14:221 [PMID:22298048].

Added note – you’ll see I have been reading a lot about collagen export recently.

One more to come soon.

F1000 evaluation

I have evaluated the following article for Faculty of 1000.

Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat.
Kung LF, Pagant S, Futai E, D’Arcangelo JG, Buchanan R, Dittmar JC, Reid RJ, Rothstein R, Hamamoto S, Snapp EL, Schekman R, Miller EA.
EMBO Journal
DOI 10.1038/emboj.2011.444
PMID 22157747

This is a very nice set of data that show convincingly that Sec16 impacts on the Sar1-GTPase cycle during COPII coat assembly at the endoplasmic reticulum (ER) membrane. There are considerable nuances within the data set likely to be of major interest to COPII aficionados but, overall, this work gives some key insight into the control of vesicle formation by the COPII coat. The [
http://f1000.com/13567956#eval14951056
Jon Audhya] evaluation describes this paper perfectly.

Additional comment: I don’t post the evaluation from Jon Audhya here. Jon highlights how this paper provides core mechanistic insight into COPII-dependent secretion through coupling the function of Sec16 to that of Sar1. This provides another piece of the puzzle in our understanding of Sec16 function. Bottom line: it is an interesting paper for those of us in this area. Read it!

Liz Miller also has a very intriguing Science paper recently published which I will comment on here once I have had time to read it properly.