Sec16 Determines the Size and Functioning of the Golgi in the Protist Parasite,Trypanosoma brucei

The Sec16 homologue in Trypanosoma brucei has been identified and characterized. TbSec16 colocalizes with COPII components at the single endoplasmic reticulum exit site (ERES), which is next to the single Golgi stack in the insect (procyclic) form of this organism. Depletion of TbSec16 reduces the size of the ERES and the Golgi, and slows growth and transport of a secretory marker to the cell surface; conversely, overexpression of TbSec16 increases the size of the ERES and Golgi but has no effect on growth or secretion. Together these data suggest that TbSec16 regulates the size of the ERES and Golgi and this size is set for optimal growth of the organism.      

ER exit sites - Localization and control of COPII vesicle formation

The first membrane trafficking step in the biosynthetic secretory pathway, the export of proteins and lipids from the endoplasmic reticulum (ER), is mediated by COPII-coated vesicles. In mammalian cells, COPII vesicle budding occurs at specialized sites on the ER, the so-called transitional ER (tER). Here, we discuss aspects of the formation and maintenance of these sites, the mechanisms by which cargo becomes segregated within them, and the propagation of ER exit sites (ERES) during cell division. All of these features are inherently linked to the formation, maintenance and function of the Golgi apparatus underlining the importance of ERES to Golgi function and more widely in terms of intracellular organization and cellular function.     

Golgi Bypass: Skirting Around the Heart of Classical Secretion

Classical secretion consists of the delivery of transmembrane and soluble proteins to the plasma membrane and the extracellular medium, respectively, and is mediated by the organelles of the secretory pathway, the Endoplasmic Reticulum (ER), the ER exit sites, and the Golgi, as described by the Nobel Prize winner George Palade ( Palade 1975). At the center of this transport route, the Golgi stack has a major role in modifying, processing, sorting, and dispatching newly synthesized proteins to their final destinations. More recently, however, it has become clear that an increasing number of transmembrane proteins reach the plasma membrane unconventionally, either by exiting the ER in non-COPII vesicles or by bypassing the Golgi. Here, we discuss the evidence for Golgi bypass and the possible physiological benefits of it. Intriguingly, at least during Drosophila development, Golgi bypass seems to be mediated by a Golgi protein, dGRASP, which is found ectopically localized to the plasma membrane.The secretion of signal peptide-containing and transmembrane proteins through the cellular organelles that form the secretory pathway has been very well characterized over the years (Rothman 1994; Lee et al. 2004). During their translation, signal peptide-containing proteins are specifically recognized in the cytoplasm by the signal recognition particle and localize to the ER by virtue of the SRP binding its receptor (Nagai et al. 2003; Osborne et al. 2005). Other transmembrane proteins are embedded in the ER membrane by a posttranslational mechanism called C-tail anchoring by the GET complex (Schuldiner et al. 2008). Following transfer into or across the ER membrane, nascent proteins undergo folding, oligomerization, and addition of oligosaccharide chains followed by exit via specialized landmarks, known as ER exit sites (ERES) in mammalian cells and transitional ER (tER) sites in yeast and Drosophila. Both sites are characterized by the presence of cargo-containing coat protein complex II (COPII)-coated vesicles (Bonifacino and Glick 2004; Lee et al. 2004). Thereafter, most proteins are transported through the Golgi (in a manner that is still very much debated) before reaching their final destination, such as the plasma membrane for many transmembrane proteins and the extracellular medium for secreted proteins (Mellman and Warren 2000) (Fig. 1, red arrows).Open in a separate windowFigure 1.Classical trafficking, from the ER to the Golgi to the plasma membrane, is represented by the red arrows. A cargo protein can exit from an ERES in close proximity to the cis-Golgi (route 1a) or a peripheral ERES (route 1b), but irrespective of its ER exit, this protein follows a distinct pathway through the Golgi to the plasma membrane. This pathway is dependent on known SNARE proteins, NSF and SNAPs. As proteins pass from the ER and through the Golgi, their ER-derived high mannose oligosaccharides are modified by addition of complex sugars rendering these proteins EndoH-resistant. BFA treatment or loss of function of intra-Golgi SNAREs would lead to the retention of these proteins in the ER or Golgi and their diminished presence at the plasma membrane.Potential routes for Golgi bypass are represented by blue arrows. Like classical cargo proteins, Golgi bypass cargoes may exit from an ERES near the cis-Golgi (routes 2a,c) or a peripheral ERES (route 2b). However, the immediate fate of these proteins deviates from the classical pathway. A protein following route 2a (from an ERES near the cis-Golgi) or 2b (from a peripheral ERES) would traffic on ER-derived transport intermediates directly to the plasma membrane, routes perhaps taken by CD45 or αPS1. This route would require a specific set of SNAREs, yet to be identified. As these proteins do not pass through the Golgi stack, their high mannose N-glycans remain sensitive to EndoH. These pathways are also revealed by blocking passage through the Golgi either by the application of BFA, or by the loss of function of intra-Golgi SNAREs, (e.g., Syntaxin 5), and observing their continued transport to the plasma membrane. Proteins that follow route 2c would bypass the Golgi stack via an endosomal intermediate, which would facilitate their delivery to the plasma membrane via conventional endosomal fusion machinery. In the case of CFTR, its exit from the ER may occur from either ERES location to the TGN or endosomes. If it is directly delivered to endosomes, it is likely recycled back to the TGN in which the observed oligosaccharide modifications take place before reaching the plasma membrane.More recently, however, several examples of protein trafficking that deviate from this dogma have been discovered. First, an increasing number of cytoplasmic proteins (such as IL-1β, FGF2, MIF, and AcbA/Acb1) that do not harbor a signal peptide are found in the extracellular medium, and these display a wide range of critical activities. This “cytoplasmic protein unconventional secretion” has been extensively discussed elsewhere (Nickel and Seedorf 2008; Nickel and Rabouille 2009) and will not be covered in this volume, except for a brief note toward the end. Second, a small subset of proteins does not exit the ER by virtue of classical COPII-coated vesicles. Third, a few transmembrane proteins have been shown to reach the plasma membrane, bypassing the Golgi, which is the focus of this article.Why some proteins follow an unconventional route of secretion is intriguing but on the whole largely unknown. Through evolution, the cell has segregated processes within membrane compartments to maintain and optimize cellular functions. Why would mechanisms evolve to traffic a subset of proteins via unconventional routes? In this article, we discuss examples of Golgi bypass as well as outline why and how some proteins escape the conventional secretory pathway.     

The chaperone Chs7 forms a stable complex with Chs3 and promotes its activity at the cell surface

The polytopic yeast protein Chs3 (chitin synthase III) relies on a dedicated membrane‐localized chaperone, Chs7, for its folding and expression at the cell surface. In the absence of Chs7, Chs3 forms high molecular weight aggregates and is retained in the endoplasmic reticulum (ER). Chs7 was reported to be an ER resident protein, but its role in Chs3 folding and transport was not well characterized. Here, we show that Chs7 itself exits the ER and localizes with Chs3 at the bud neck and intracellular compartments. We identified mutations in the Chs7 C‐terminal cytosolic domain that do not affect its chaperone function, but cause it to dissociate from Chs3 at a post‐ER transport step. Mutations that prevent the continued association of Chs7 with Chs3 do not block delivery of Chs3 to the cell surface, but dramatically reduce its catalytic activity. This suggests that Chs7 engages in functionally distinct interactions with Chs3 to first promote its folding and ER exit, and subsequently to regulate its activity at the plasma membrane.      

Kinesin-1 (uKHC/KIF5B) is required for bidirectional motility of ER exit sites and efficient ER-to-Golgi transport

Transport of proteins and lipids between intracellular compartments is fundamental to the organization and function of eukaryotic cells. The efficiency of this process is greatly enhanced through coupling of membranes to microtubules. This serves two functions, organelle positioning and vesicular transport. In this study, we show that in addition to the well-known role for the minus-end motor dynein in endoplasmic reticulum (ER)-to-Golgi transport, the plus-end-directed motor kinesin-1 is involved in positioning coat protein II-coated ER exit sites (ERES) in cells as well as the formation of transport carriers and their movement to the Golgi. Using two-dimensional Gaussian fitting to determine their location at high spatial resolution, we show that ERES undergo short-range bidirectional movements. Bidirectionality depends on both kinesin-1 and dynein. Suppression of kinesin-1 (KIF5B) also inhibits ER-to-Golgi transport and affects the morphology of ER-to-Golgi transport carriers. Furthermore, we show that suppression of dynein heavy chain expression increases the range of movement of ERES, suggesting that dynein might anchor ERES, or the ER itself, to microtubules. These data implicate kinesin-1 in the spatial organization of the ER/Golgi interface as well as in traffic outside the ER.     

Leucine-rich repeat kinase 2 regulates Sec16A at ER exit sites to allow ER–Golgi export

Leucine-rich repeat kinase 2 (LRRK2) has been associated with Parkinson’s disease (PD) and other disorders. However, its normal physiological functions and pathogenic properties remain elusive. Here we show that LRRK2 regulates the anterograde ER–Golgi transport through anchoring Sec16A at the endoplasmic reticulum exit sites (ERES). LRRK2 interacted and co-localized with Sec16A, a key protein in the formation of ERES. Lrrk2 depletion caused a dispersion of Sec16A from ERES and impaired ER export. In neurons, LRRK2 and Sec16A showed extensive co-localization at the dendritic ERES (dERES) that locally regulate the transport of proteins to the dendritic spines. A loss of Lrrk2 affected the association of Sec16A with dERES and impaired the activity-dependent targeting of glutamate receptors onto the cell/synapse surface. Furthermore, the PD-related LRRK2 R1441C missense mutation in the GTPase domain interfered with the interaction of LRRK2 with Sec16A and also affected ER–Golgi transport, while LRRK2 kinase activity was not required for these functions. Therefore, our findings reveal a new physiological function of LRRK2 in ER–Golgi transport, suggesting ERES dysfunction may contribute to the pathogenesis of PD.     

Control of MHC class I traffic from the endoplasmic reticulum by cellular chaperones and viral anti-chaperones

MHC class I molecules assemble with peptides in the endoplasmic reticulum (ER). To ensure that only peptide-loaded MHC molecules leave the ER, empty molecules are retained by ER-resident chaperones, most notably the MHC-specific tapasin. ER exit of class I MHC is also controlled by viruses, but for the opposite purpose of preventing peptide presentation to T cells. Interestingly, some viral proteins are able to retain MHC class I molecules in the ER despite being transported. By contrast, other viral proteins exit the ER only upon binding to class I MHC, thereby rerouting newly synthesized class I molecules to intracellular sites of proteolysis. Thus, immune escape can be achieved by reversing, inhibiting or redirecting the chaperone-assisted MHC class I folding, assembly and intracellular transport.     

ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I-mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.     

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR

Induction of endoplasmic reticulum (ER)‐to‐Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)‐mediated, Golgi‐independent unconventional cell‐surface trafficking of the folding‐deficient ΔF508‐cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation‐dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508‐CFTR, such as induction of ER‐to‐Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C‐terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation‐mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation‐inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER‐retained ΔF508‐CFTR and mediates the ER stress‐induced unconventional secretion pathway.      

A New Role for Annexin A11 in the Early Secretory Pathway via Stabilizing Sec31A Protein at the Endoplasmic Reticulum Exit Sites (ERES)

Exit of cargo molecules from the endoplasmic reticulum (ER) for transport to the Golgi is the initial step in intracellular vesicular trafficking. The coat protein complex II (COPII) machinery is recruited to specialized regions of the ER, called ER exit sites (ERES), where it plays a central role in the early secretory pathway. It has been known for more than two decades that calcium is an essential factor in vesicle trafficking from the ER to Golgi apparatus. However, the role of calcium in the early secretory pathway is complicated and poorly understood. We and others previously identified Sec31A, an outer cage component of COPII, as an interacting protein for the penta-EF-hand calcium-binding protein ALG-2. In this study, we show that another calcium-binding protein, annexin A11 (AnxA11), physically associates with Sec31A by the adaptor function of ALG-2. Depletion of AnxA11 or ALG-2 decreases the population of Sec31A that is stably associated with the ERES and causes scattering of juxtanuclear ERES to the cell periphery. The synchronous ER-to-Golgi transport of transmembrane cargoes is accelerated in AnxA11- or ALG-2-knockdown cells. These findings suggest that AnxA11 maintains architectural and functional features of the ERES by coordinating with ALG-2 to stabilize Sec31A at the ERES.     

Streamlined Architecture and Glycosylphosphatidylinositol-dependent Trafficking in the Early Secretory Pathway of African Trypanosomes

The variant surface glycoprotein (VSG) of bloodstream form Trypanosoma brucei (Tb) is a critical virulence factor. The VSG glycosylphosphatidylinositol (GPI)-anchor strongly influences passage through the early secretory pathway. Using a dominant-negative mutation of TbSar1, we show that endoplasmic reticulum (ER) exit of secretory cargo in trypanosomes is dependent on the coat protein complex II (COPII) machinery. Trypanosomes have two orthologues each of the Sec23 and Sec24 COPII subunits, which form specific heterodimeric pairs: TbSec23.1/TbSec24.2 and TbSec23.2/TbSec24.1. RNA interference silencing of each subunit is lethal but has minimal effects on trafficking of soluble and transmembrane proteins. However, silencing of the TbSec23.2/TbSec24.1 pair selectively impairs ER exit of GPI-anchored cargo. All four subunits colocalize to one or two ER exit sites (ERES), in close alignment with the postnuclear flagellar adherence zone (FAZ), and closely juxtaposed to corresponding Golgi clusters. These ERES are nucleated on the FAZ-associated ER. The Golgi matrix protein Tb Golgi reassembly stacking protein defines a region between the ERES and Golgi, suggesting a possible structural role in the ERES:Golgi junction. Our results confirm a selective mechanism for GPI-anchored cargo loading into COPII vesicles and a remarkable degree of streamlining in the early secretory pathway. This unusual architecture probably maximizes efficiency of VSG transport and fidelity in organellar segregation during cytokinesis.     

Mechanisms of the anterograde trafficking of GPCRs: Regulation of AT1R transport by interacting proteins and motifs

Anterograde cell surface transport of nascent G protein‐coupled receptors (GPCRs) en route from the endoplasmic reticulum (ER) through the Golgi apparatus represents a crucial checkpoint to control the amount of the receptors at the functional destination and the strength of receptor activation‐elicited cellular responses. However, as compared with extensively studied internalization and recycling processes, the molecular mechanisms of cell surface trafficking of GPCRs are relatively less defined. Here, we will review the current advances in understanding the ER‐Golgi‐cell surface transport of GPCRs and use angiotensin II type 1 receptor as a representative GPCR to discuss emerging roles of receptor‐interacting proteins and specific motifs embedded within the receptors in controlling the forward traffic of GPCRs along the biosynthetic pathway.      

Mutant SOD1 inhibits ER‐Golgi transport in amyotrophic lateral sclerosis

Cu/Zn‐superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER‐Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER‐Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER‐Golgi transport by over‐expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER‐Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

     

Syntaxin 17 cycles between the ER and ERGIC and is required to maintain the architecture of ERGIC and Golgi

Background information. Syntaxin 17 is a SNARE (soluble N‐ethylmaleimide‐sensitive‐factor‐attachment protein receptor) protein that predominantly localizes to the ER (endoplasmic reticulum) and to some extent in the ERGIC (ER—Golgi intermediate compartment). Syntaxin 17 has been suggested to function as a receptor at the ER membrane that mediates trafficking between the ER and post‐ER compartments. It has a unique 33 amino acid luminal tail whose function is not known. Here we have investigated the structural requirements for localization of syntaxin 17 to the ERGIC and its role in trafficking. Results. Deletion analysis showed that syntaxin 17 required its cytoplasmic domain to exit the ER and localize to the ERGIC. Mutation of a conserved tyrosine residue in the cytoplasmic domain resulted in reduced localization of syntaxin 17 in the ERGIC and ER‐exit sites, suggesting the presence of a tyrosine‐based ER export motif. Syntaxin 17 also required its C‐terminal tail to localize to the ERES (ER exit sites) and ERGIC. Knockdown of syntaxin 17 destabilized the ERGIC organization and also caused fragmentation of the Golgi complex. Syntaxin 17 showed direct interaction with transmembrane proteins p23 and p25 (cargo receptors that cycle between the ER and Golgi) with the help of its C‐terminal tail. Overexpression of syntaxin 17 redistributed β‐COP (β‐coatomer protein) which required its C‐terminal tail. Overexpression of syntaxin 17 also blocked the anterograde transport of VSVG (vesicular stomatitis virus G‐protein) in the ERGIC. Conclusions. We show that syntaxin 17 has a tyrosine‐based motif which is required for its incorporation into COPII (coatomer protein II) vesicles, exit from the ER and localization to the ERGIC. Our results suggest that syntaxin 17 cycles between the ER and ERGIC through classical trafficking pathways involving COPII and COPI (coatomer protein I) vesicles, which requires its unique C‐terminal tail. We also show that syntaxin 17 is essential for maintaining the architecture of ERGIC and Golgi.     

Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei

The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre‐cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post‐mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post‐mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol‐anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans‐face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi.      

Export of cyst wall material and Golgi organelle neogenesis in Giardia lamblia depend on endoplasmic reticulum exit sites

Giardia lamblia parasitism accounts for the majority of cases of parasitic diarrheal disease, making this flagellated eukaryote the most successful intestinal parasite worldwide. This organism has undergone secondary reduction/elimination of entire organelle systems such as mitochondria and Golgi. However, trophozoite to cyst differentiation (encystation) requires neogenesis of Golgi‐like secretory organelles named encystation‐specific vesicles (ESVs), which traffic, modify and partition cyst wall proteins produced exclusively during encystation. In this work we ask whether neogenesis of Golgi‐related ESVs during G. lamblia differentiation, similarly to Golgi biogenesis in more complex eukaryotes, requires the maintenance of distinct COPII‐associated endoplasmic reticulum (ER) subdomains in the form of ER exit sites (ERES) and whether ERES are also present in non‐differentiating trophozoites. To address this question, we identified conserved COPII components in G. lamblia cells and determined their localization, quantity and dynamics at distinct ERES domains in vegetative and differentiating trophozoites. Analogous to ERES and Golgi biogenesis, these domains were closely associated to early stages ofnewly generated ESV. Ectopic expression of non‐functional Sar1 GTPase variants caused ERES collapse and, consequently, ESV ablation, leading to impaired parasite differentiation. Thus, our data show how ERES domains remain conserved in G. lamblia despite elimination of steady‐state Golgi. Furthermore, the fundamental eukaryotic principle of ERES to Golgi/Golgi‐like compartment correspondence holds true in differentiating Giardia presenting streamlined machinery for secretory organelle biogenesis and protein trafficking. However, in the Golgi‐less trophozoites ERES exist as stable ER subdomains, likely as the sole sorting centres for secretory traffic.     

Adaptor functions of the Ca2+-binding protein ALG-2 in protein transport from the endoplasmic reticulum

Apoptosis-linked gene 2 (ALG-2) is a Ca²⁺-binding protein with five repetitive EF-hand motifs, named penta-EF-hand (PEF) domain. It interacts with various target proteins and functions as a Ca²⁺-dependent adaptor in diverse cellular activities. In the cytoplasm, ALG-2 is predominantly localized to a specialized region of the endoplasmic reticulum (ER), called the ER exit site (ERES), through its interaction with Sec31A. Sec31A is an outer coat protein of coat protein complex II (COPII) and is recruited from the cytosol to the ERES to form COPII-coated transport vesicles. I will overview current knowledge of the physiological significance of ALG-2 in regulating ERES localization of Sec31A and the following adaptor functions of ALG-2, including bridging Sec31A and annexin A11 to stabilize Sec31A at the ERES, polymerizing the Trk-fused gene (TFG) product, and linking MAPK1-interacting and spindle stabilizing (MISS)-like (MISSL) and microtubule-associated protein 1B (MAP1B) to promote anterograde transport from the ER.     

Calreticulin‐dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin‐deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub‐optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER–Golgi intermediate compartment (ERGIC) and the cis‐Golgi, together with sub‐optimally loaded class I molecules. Calreticulin that lacks its C‐terminal KDEL retrieval sequence assembles with the peptide‐loading complex but neither retrieves sub‐optimally loaded class I molecules from the cis‐Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.     

Plant Sar1 isoforms with near-identical protein sequences exhibit different localisations and effects on secretion

In plants, differentiation of subdomains of the endoplasmic reticulum (ER) dedicated to protein export, the ER export sites (ERES), is influenced by the type of export-competent membrane cargo to be delivered to the Golgi. This raises a fundamental biological question: is the formation of transport intermediates at the ER for trafficking to the Golgi always regulated in the same manner? To test this, we followed the distribution and activity of two plant Sar1 isoforms. Sar1 is the small GTPase that regulates assembly of COPII (coat protein complex II) on carriers that transport secretory cargo from ER to Golgi. We show that, in contrast to a tobacco Sar1 isoform, the two Arabidopsis Sar1 GTPases were localised at ERES, independently of co-expression of Golgi-destined membrane cargo in tobacco cells. Although both isoforms labelled ERES, one was found to partition with the membrane fraction to a greater extent. The different distribution of fluorescent fusions of the two isoforms was influenced by the nature of an amino acid residue at the C-terminus of the protein, suggesting that the requirements for membrane association of the two GTPases are not equal. Furthermore, functional analyses based on the secretion of the bulk flow marker α-amylase indicated that over-expression of GTP-restricted mutants of the two isoforms caused different levels of ER export inhibition. These novel results indicate a functional heterogeneity among plant Sar1 isoforms.     

Kinetically Distinct Sorting Pathways through the Golgi Exhibit Different Requirements for Arf1

To investigate the role of cytoplasmic sequences in directing transmembrane protein trafficking through the Golgi, we analyzed the sorting of VSV tsO45 G fusions with either the native G cytoplasmic domain (G) or an alternative cytoplasmic tail derived from the chicken AE1‐4 anion exchanger (G^AE). At restrictive temperature G^AE and G accumulated in the ER, and upon shifting the cells to permissive temperature both proteins folded and underwent transport through the Golgi. However, G^AE and G did not form hetero‐oligomers upon the shift to permissive temperature and they progressed through the Golgi with distinct kinetics. In addition, the transport of G through the proximal Golgi was Arf1 and COPI‐dependent, while G^AE progression through the proximal Golgi was Arf1 and COPI‐independent. Although Arf1 did not regulate the sorting of G^AE in the cis‐Golgi, Arf1 did regulate the exit of G^AE from the TGN. The trafficking of G^AE through the Golgi was similar to that of the native AE1‐4 anion exchanger, in that the progression of both proteins through the proximal Golgi was Arf1‐independent, while both required Arf1 to exit the TGN. We propose that the differential recognition of cytosolic signals in membrane‐spanning proteins by the Arf1‐dependent sorting machinery may influence the rate at which cargo progresses through the Golgi.