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Sean Munro

Publications and source records attributed to Sean Munro.

At least 19 recordsLinked to original sources
PubMed2006

Selective export of HLA-F by its cytoplasmic tail.

MHC class I molecules exit the endoplasmic reticulum (ER) by an unknown mechanism. Although a selective export mechanism has been proposed for the anterograde transport of class I, a motif responsible for export has never been identified. Although classical class I molecules lacking their cytoplasmic tail are expressed on the cell surface, we found that HLA-F was entirely dependent on its cytoplasmic tail for export from the ER. Two known export motifs were recognizable in HLA-F. A C-terminal valine residue functioned in ER export and interacted with coat complex (COP)II, while an RxR motif also played an important role in anterograde transport and bound to 14-3-3 proteins. This divergent trafficking of HLA-F implicates an alternative function for HLA-F, independent of loading with peptides in the ER.

14-3-3 Proteins↗
PubMed2006

An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility.

Small GTPases of the Arf and Rab families play key roles in the function of subcellular organelles. Each GTPase is usually found on only one compartment and, hence, they confer organelle specificity to many intracellular processes. However, there has so far been little evidence for specific GTPases present on lysosomes. Here, we report that two closely related human Arf-like GTPases, Arl8a and Arl8b (also known as Arl10b/c and Gie1/2), localise to lysosomes in mammalian cells, with the single homologue in Drosophila cells having a similar location. Conventionally, membrane binding of Arf and Arl proteins is mediated by both an N-terminal myristoyl group and an N-terminal amphipathic helix that is inserted into the lipid bilayer upon activation of the GTPase. Arl8a and Arl8b do not have N-terminal myristoylation sites, and we find that Arl8b is instead N-terminally acetylated, and an acetylated methionine is necessary for its lysosomal localization. Overexpression of Arl8a or Arl8b results in a microtubule-dependent redistribution of lysosomes towards the cell periphery. Live cell imaging shows that lysosomes move more frequently both toward and away from the cell periphery, suggesting a role for Arl8a and Arl8b as positive regulators of lysosomal transport.

ADP-Ribosylation Factors↗
PubMed2006

Nomenclature for the human Arf family of GTP-binding proteins: ARF, ARL, and SAR proteins.

The Ras superfamily is comprised of at least four large families of regulatory guanosine triphosphate-binding proteins, including the Arfs. The Arf family includes three different groups of proteins: the Arfs, Arf-like (Arls), and SARs. Several Arf family members have been very highly conserved throughout eukaryotic evolution and have orthologues in evolutionally diverse species. The different means by which Arf family members have been identified have resulted in an inconsistent and confusing array of names. This confusion is further compounded by differences in nomenclature between different species. We propose a more consistent nomenclature for the human members of the Arf family that may also serve as a guide for nomenclature in other species.

ADP-Ribosylation Factors↗
PubMed2005

Organelle identity and the signposts for membrane traffic.

Eukaryotic cells have systems of internal organelles to synthesize lipids and membrane proteins, to release secreted proteins, to take up nutrients and to degrade membrane-bound and internalized molecules. Proteins and lipids move from organelle to organelle using transport vesicles. The accuracy of this traffic depends upon organelles being correctly recognized. In general, organelles are identified by the activated GTPases and specific lipid species that they display. These short-lived determinants provide organelles with an identity that is both unique and flexible. Recent studies have helped to establish how cells maintain and restrict these determinants and explain how this system is exploited by invading pathogens.

Cell Membrane↗
PubMed2005

Mon2, a relative of large Arf exchange factors, recruits Dop1 to the Golgi apparatus.

The protein Mon2 is distantly related to the guanine nucleotide exchange factors (GEFs) that activate Arf1 on Golgi membranes. However, unlike these "large" Arf GEFs, Mon2 lacks the Sec7 domain that catalyzes nucleotide exchange on Arf1. Here we report that yeast Mon2 shares extensive homology with the noncatalytic parts of both the BIG and Golgi brefeldin A resistance factor subfamilies of Arf GEFs and is located to the trans-Golgi. Moreover, we find that Mon2 forms a complex with Dop1, a large cytoplasmic protein conserved in evolution from humans to protozoa. Deletion of Mon2 results in mislocalization of Dop1 from the Golgi and defects in cycling between endosomes and the Golgi. However, unlike Mon2, Dop1 is essential for yeast viability. A conditional allele of Dop1 shows that loss of Dop1 activity not only affects endosome to Golgi transport but also causes a severe perturbation of the organization of the endoplasmic reticulum. Thus, it appears that Dop1 plays a widespread role in membrane organization, and Mon2 acts as a scaffold to recruit the Golgi-localized pool of Dop1.

Alleles↗
PubMed2005

The exocyst component Sec5 is present on endocytic vesicles in the oocyte of Drosophila melanogaster.

The exocyst is an octameric complex required for polarized secretion. Some components of the exocyst are found on the plasma membrane, whereas others are recruited to Golgi membranes, suggesting that exocyst assembly tethers vesicles to their site of fusion. We have found that in Drosophila melanogaster oocytes the majority of the exocyst component Sec5 is unexpectedly present in clathrin-coated pits and vesicles at the plasma membrane. In oocytes, the major substrate for clathrin-dependent endocytosis is the vitellogenin receptor Yolkless. A truncation mutant of Sec5 (sec5(E13)) allows the formation of normally sized oocytes but with greatly reduced yolk uptake. We find that in sec5(E13) oocytes Yolkless accumulates aberrantly in late endocytic compartments, indicating a defect in the endocytic cycling of the receptor. An analogous truncation of the yeast SEC5 gene results in normal secretion but a temperature-sensitive defect in endocytic recycling. Thus, the exocyst may act in both Golgi to plasma membrane traffic and endocytic cycling, and hence in oocytes is recruited to clathrin-coated pits to facilitate the rapid recycling of Yolkless.

Animals↗
PubMed2005

The Golgi apparatus: defining the identity of Golgi membranes.

The Golgi apparatus is a stack of compartments that serves as a central junction for membrane traffic, with carriers moving through the stack as well as arriving from, and departing toward, many other destinations in the cell. This requires that the different compartments in the Golgi recruit from the cytosol a distinct set of proteins to mediate accurate membrane traffic. This recruitment appears to reflect recognition of small GTPases of the Rab and Arf family, or of lipid species such as PtdIns(4)P and diacylglycerol, which provide a unique "identity" for each compartment. Recent work is starting to reveal the mechanisms by which these labile landmarks are generated in a spatially restricted manner on specific parts of the Golgi.

Animals↗
PubMed2004

The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi.

Rud3p is a coiled-coil protein of the yeast cis-Golgi. We find that Rud3p is localized to the Golgi via a COOH-terminal domain that is distantly related to the GRIP domain that recruits several coiled-coil proteins to the trans-Golgi by binding the small Arf-like GTPase Arl1p. In contrast, Rud3p binds to the GTPase Arf1p via this COOH-terminal "GRIP-related Arf-binding" (GRAB) domain. Deletion of RUD3 is lethal in the absence of the Golgi GTPase Ypt6p, and a screen of other mutants showing a similar genetic interaction revealed that Golgi targeting of Rud3p also requires Erv14p, a cargo receptor that cycles between the endoplasmic reticulum and Golgi. The one human protein with a GRAB domain, GMAP-210 (CEV14/Trip11/Trip230), is known to be on the cis-Golgi, but the COOH-terminal region that contains the GRAB domain has been reported to bind to centrosomes and gamma-tubulin (Rios, R.M, A. Sanchis, A.M. Tassin, C. Fedriani, and M. Bornens. 2004. Cell. 118:323-335). In contrast, we find that this region binds to the Golgi in a GRAB domain-dependent manner, suggesting that GMAP-210 may not link the Golgi to gamma-tubulin and centrosomes.

ADP-Ribosylation Factor 1↗
PubMed2004

Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p.

The GTPase Arl3p is required to recruit a second GTPase, Arl1p, to the Golgi in Saccharomyces cerevisiae. Arl1p binds to the GRIP domain, which is present in a number of long coiled-coil proteins or 'golgins'. Here we show that Arl3p is not myristoylated like most members of the Arf family, but is instead amino-terminally acetylated by the NatC complex. Targeting of Arl3p also requires a Golgi membrane protein Sys1p. The human homologues of Arl3p (Arf-related protein 1 (ARFRP1)) and Sys1p (hSys1) can be isolated in a complex after chemical cross-linking. This suggests that the targeting of ARFRP1/Arl3p to the Golgi is mediated by a direct interaction between its acetylated N terminus and Sys1p/hSys1.

ADP-Ribosylation Factor 1↗
PubMed2004

Global mapping of the yeast genetic interaction network.

A genetic interaction network containing approximately 1000 genes and approximately 4000 interactions was mapped by crossing mutations in 132 different query genes into a set of approximately 4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects. Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway. The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions. Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.

Amino Acid Sequence↗
PubMed2004

Organelle identity and the organization of membrane traffic.

Generating and maintaining features that distinguish one organelle from another is essential for accurate membrane traffic. Recent work has revealed that organelles express 'identity' by the local generation of activated GTP-binding proteins and lipid species. These recruiting determinants are then recognized by cytosolic proteins that facilitate the formation and delivery of vesicles at the correct compartment.

Animals↗
PubMed2003

Lipid rafts: elusive or illusive?

There has been considerable recent interest in the possibility that the plasma membrane contains lipid "rafts," microdomains enriched in cholesterol and sphingolipids. It has been suggested that such rafts could play an important role in many cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry. However, rafts have proven difficult to visualize in living cells. Most of the evidence for their existence and function relies on indirect methods such as detergent extraction, and a number of recent studies have revealed possible problems with these methods. Direct studies of the distribution of raft components in living cells have not yet reached a consensus on the size or even the presence of these microdomains, and hence it seems that a definitive proof of raft existence has yet to be obtained.

Animals↗
PubMed2003

Long coiled-coil proteins and membrane traffic.

Protein transport between organelles is mediated by vesicles which must accurately dock and fuse with appropriate compartments. Over the past several years a large number of long coiled-coil proteins have been identified on the Golgi and on endosomes, mostly as auto-antigens in autoimmune disorders. Based on their restricted intracellular distributions and their predicted rod-like structure, these proteins have been proposed to play a role in tethering vesicles to target organelles prior to fusion. However, such proteins may also play a structural role, for example as components of a Golgi matrix, or as scaffolds for the assembly of other factors important for fusion. This review will examine what is known about the function of these large coiled-coil proteins in membrane traffic.

Animals↗
PubMed2003

An N-acetylglucosaminyltransferase of the Golgi apparatus of the yeast Saccharomyces cerevisiae that can modify N-linked glycans.

The yeast Saccharomyces cerevisiae is widely regarded as being only capable of producing N-linked glycans with high-mannose structures. To investigate the glycan structures made in different mutant strains, we made use of a reporter protein consisting of a version of hen egg lysozyme that contains a single site for N-linked glycosylation. Mass spectrometry analysis of the attached glycans revealed that a large proportion contained an unexpected extra mass corresponding to a single N-acetylhexosamine residue. In addition, the glycosylated lysozyme was recognized by an N-acetylglucosamine specific lectin. The genome of S. cerevisiae contains an uncharacterized open reading frame, YOR320c, that is related to a known N-acetylglucosaminyltransferase. Deletion of this ORF resulted in the disappearance of the extra mass on the N-linked glycans and loss of lectin binding. We show that the protein encoded by YOR320c (which we term Gnt1p) is localized to the Golgi apparatus and has GlcNAc-transferase activity in vitro. The physiological role of Gnt1p is unclear because mutants lacking the protein show no obvious growth or cell wall defects. Nonetheless, these results indicate that heterologous glycoproteins expressed in yeast can receive N-glycans with structures other than high mannose. In addition, they indicate that the lumen of the yeast Golgi contains UDP-GlcNAc, which may facilitate reconstitution of higher eukaryotic N-glycan processing.

Amino Acid Sequence↗
PubMed2003

The ARF-like GTPases Arl1p and Arl3p act in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus.

The ARLs are a diverse family of GTPases that are related to ADP-ribosylation factors (ARFs), but whose function is poorly understood. There are at least ten ARLs in humans, two of which have homologs in the yeast Saccharomyces cerevisiae (ARL1/Arl1p and ARFRP1/Arl3p). The function of ARFRP1 is unknown, but mammalian ARL1 has recently been found to interact with a number of effectors including the GRIP domain that is present in a family of Golgi-localized long coiled-coil proteins. We find that in yeast, the intracellular targeting of Imh1p, the only yeast GRIP domain protein, is dependent on both Arl1p and Arl3p, but not on the ARF proteins. A recombinant form of the Imh1p GRIP domain binds to Arl1p in a GTP-dependent manner, but not to Arl3p. Yeast also contain a relative of SCOCO, a protein proposed to bind human ARL1, but this yeast protein, Slo1p, appears to bind Arl3p rather than Arl1p in vitro. However, Imh1p is not the sole effector of Arl1p since affinity chromatography of cytosol with immobilized Arl1p:GTP revealed an interaction with the GARP/VFT complex that is thought to act in the tethering of vesicles to the Golgi apparatus. Finally, we find that Arl3p is required in vivo for the targeting of Arl1p, explaining its requirement for the normal distribution of Imh1p.

ADP-Ribosylation Factors↗
PubMed2003

Structural basis for Arl1-dependent targeting of homodimeric GRIP domains to the Golgi apparatus.

Golgins are large coiled-coil proteins that play a role in Golgi structure and vesicle traffic. The Arf-like GTPase Arl1 regulates the translocation of GRIP domain-containing golgins to Golgi membranes. We report here the 1.7 A resolution structure of human Arl1-GTP in a complex with the GRIP domain of golgin-245. The structure reveals that the GRIP domain consists of an S-shaped arrangement of three helices. The domain forms a homodimer that binds two Arl1-GTPs using two helices from each monomer. The structure is consistent with golgin-245 forming parallel coiled-coils and suggests how Arl1-GTP/GRIP complexes interact with Golgi membranes via the N termini of Arl1-GTP and the C-terminal tails of the GRIP domains. In cells, bivalent association with Arl1-GTP would increase residence time of the golgins on Golgi membranes. Despite no conservation of sequence, topology, or even helical direction, several other effectors form similar interactions with small GTPases via a pair of alpha helices, suggesting a common structural basis for effector recognition.

ADP-Ribosylation Factors↗
PubMed2002

The components of the Saccharomyces cerevisiae mannosyltransferase complex M-Pol I have distinct functions in mannan synthesis.

The yeast Saccharomyces cerevisiae processes N-linked glycans in the Golgi apparatus in two different ways. Whereas most of the proteins of internal membranes receive a simple core-type structure, a long branched polymer termed mannan is attached to the glycans of many of the proteins destined for the cell wall. The first step in mannan synthesis is the initiation and extension of an alpha-1,6-linked polymannose backbone. This requires the sequential action of two enzyme complexes, mannan polymerases (M-Pol) I and II. M-Pol I contains the proteins Mnn9p and Van1p, although the stoichiometry and individual contributions to enzyme action are unclear. We report here that the two proteins are each present as a single copy in the complex. Both proteins contain a DXD motif found in the active site of many glycosyltransferases, and mutations in this motif in Mnn9p or Van1p reveal that both proteins contribute to mannose polymerization. However, the effects of these mutations on both the in vivo and in vitro activity are distinct, suggesting that the two proteins may have different roles in the complex. Finally, we show that a simple glycoprotein based on hen egg lysozyme can be used as a substrate for modification by purified M-Pol I in vitro.

Amino Acid Motifs↗