A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus

SAR1, a gene that has been isolated as a multicopy suppressor of the yeast ER-Golgi transport mutant sec12, encodes a novel GTP-binding protein. Its nucleotide sequence predicts a 21-kD polypeptide that contains amino acid sequences highly homologous to GTP-binding domains of many ras-related proteins. Gene disruption experiments show that SAR1 is essential for cell growth. To test its function further, SAR1 has been placed under control of the GAL1 promoter and introduced into a haploid cell that had its chromosomal SAR1 copy disrupted. This mutant grows normally in galactose medium but arrests growth 12-15 h after transfer to glucose medium. At the same time, mutant cells accumulate ER precursor forms of a secretory pheromone, alpha-mating factor, and a vacuolar enzyme, carboxypeptidase Y. We propose that Sec12p and Sarlp collaborate in directing ER-Golgi protein transport.     

The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the ras-related protein Rho1p is essentially the only target for ADP-ribosylation by exoenzyme C3 of Clostridium botulinum. Using C3 to detect Rho1p in subcellular fractions, Rho1p was found primarily in the 10,000 g pellet (P2) containing large organelles; small amounts also were detected in the 100,000 g pellet (P3), and cytosol. When P2 organelles were separated in sucrose density gradients Rho1p comigrated with the Kex-2 activity, a late Golgi marker. Rho1p distribution was shifted from P2 to P3 in several mutants that accumulate post-Golgi vesicles. Rho1p comigrated with post-Golgi transport vesicles during fractionation of P3 organelles from wild-type or sec6 cells. Vesicles containing Rho1p were of the same size but different density than those bearing Sec4p, a ras-related protein located both on post-Golgi vesicles and the plasma membrane. Immunofluorescence microscopy detected Rho1p as a punctate pattern, with signal concentrated towards the cell periphery and in the bud. Thus, in S. cerevisiae Rho1p resides primarily in the Golgi apparatus, and also in vesicles that are likely to be early post-Golgi vesicles.     

Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p

A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Delta strain displays a growth defect on synthetic medium at 37 degrees C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Delta cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Delta cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.     

Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport

In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.     

TBCCD1, a new centrosomal protein,is required for centrosome and Golgi apparatus positioning

In animal cells the centrosome is positioned at the cell centre in close association with the nucleus. The mechanisms responsible for this are not completely understood. Here, we report the first characterization of human TBCC‐domain containing 1 (TBCCD1), a protein related to tubulin cofactor C. TBCCD1 localizes at the centrosome and at the spindle midzone, midbody and basal bodies of primary and motile cilia. Knockdown of TBCCD1 in RPE‐1 cells caused the dissociation of the centrosome from the nucleus and disorganization of the Golgi apparatus. TBCCD1‐depleted cells are larger, less efficient in primary cilia assembly and their migration is slower in wound‐healing assays. However, the major microtubule‐nucleating activity of the centrosome is not affected by TBCCD1 silencing. We propose that TBCCD1 is a key regulator of centrosome positioning and consequently of internal cell organization.     

The yeast p24 complex is required for the formation of COPI retrograde transport vesicles from the Golgi apparatus

The p24 family members are transmembrane proteins assembled into heteromeric complexes that continuously cycle between the ER and the Golgi apparatus. These cargo proteins were assumed to play a structural role in COPI budding because of their major presence in mammalian COPI vesicles. However, this putative function has not been proved conclusively so far. Furthermore, deletion of all eight yeast p24 family members does not produce severe transport phenotypes, suggesting that the p24 complex is not essential for COPI function. In this paper we provide direct evidence that the yeast p24 complex plays an active role in retrograde transport from Golgi to ER by facilitating the formation of COPI-coated vesicles. Therefore, our results demonstrate that p24 proteins are important for vesicle formation instead of simply being a passive traveler, supporting the model in which cargo together with a small GTPase of the ARF superfamily and coat subunits act as primer for vesicle formation.     

RAF1-activated MEK1 is found on the Golgi apparatus in late prophase and is required for Golgi complex fragmentation in mitosis

Amitotically activated mitogen-activated protein kinase 1 (MEK1) fragments the pericentriolar Golgi stacks in mammalian cells. We show that activated MEK1 is found on the Golgi apparatus in late prophase. The fragmented and dispersed Golgi membranes in prometaphase and later stages of mitosis do not contain activated MEK1. MEK1-dependent Golgi complex fragmentation is through activation by RAF1 and not MEK1 kinase 1. We propose that a RAF1-dependent activation of MEK1 and its presence on the Golgi apparatus in late prophase is required for Golgi complex fragmentation.     

The NOG1 GTP-binding protein is required for biogenesis of the 60 S ribosomal subunit

NOG1 is a nucleolar GTP-binding protein present in eukaryotes ranging from trypanosomes to humans. In this report we demonstrate that NOG1 is functionally linked to ribosome biogenesis. In sucrose density gradients Trypanosoma brucei NOG1 co-sediments with 60 S ribosomal subunits but not with monosomes. 60 S precursor RNAs are co-precipitated with NOG1. Together with the nucleolar localization of NOG1, these data indicate that NOG1 is associated with a precursor particle to the 60 S subunit. Disruption of NOG1 function through RNA interference led to a dramatic decrease in the levels of free 60 S particles and the appearance of an atypical rRNA intermediate in which ITS2 was not cleaved. Overexpression of mutant nog1 with a defect in its GTP binding motif on a wild type background caused a modest defect in 60 S biogenesis and a relative decrease in processing of the large subunit rRNAs. In contrast to the mutant protein, neither the N-terminal half of NOG1, which contains the GTP binding motifs, nor the C-terminal half of NOG1 associated with pre-ribosomal particles, although both localized to the nucleolus.     

The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly

Eukaryotic cilia are assembled via intraflagellar transport (IFT) in which large protein particles are motored along ciliary microtubules. The IFT particles are composed of at least 17 polypeptides that are thought to contain binding sites for various cargos that need to be transported from their site of synthesis in the cell body to the site of assembly in the cilium. We show here that the IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. Strong knock down of IFT20 in mammalian cells blocks ciliary assembly but does not affect Golgi structure. Moderate knockdown does not block cilia assembly but reduces the amount of polycystin-2 that is localized to the cilia. This work suggests that IFT20 functions in the delivery of ciliary membrane proteins from the Golgi complex to the cilium.     

Retrograde transport of protein toxins through the Golgi apparatus

A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.     

A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.     

The import of S-adenosylmethionine into the Golgi apparatus is required for the methylation of homogalacturonan

S-adenosylmethionine (SAM) is the substrate used in the methylation of homogalacturonan (HGA) in the Golgi apparatus. SAM is synthesized in the cytosol, but it is not currently known how it is then transported into the Golgi. In this study, we find that HGA methyltransferase is present in Golgi-enriched fractions and that its catalytic domain faces the lumen of this organelle. This suggests that SAM must be imported into the Golgi. We performed uptake experiments using [methyl-(14)C]SAM and found that SAM is incorporated into the Golgi vesicles, resulting in the methylation of polymers that are sensitive to pectinase and pectin methylesterase but not to proteases. To avoid detecting the transfer reaction, we also used [carboxyl-(14)C]SAM, the uptake of which into Golgi vesicles was found to be sensitive to temperature, detergents, and osmotic changes, and to be saturable with a K(m) of 33 microm. Double-label uptake experiments using [methyl-(3)H]SAM and [carboxyl-(14)C]SAM also revealed a time-dependent increase in the (3)H to (14)C ratio, suggesting that upon transfer of the methyl group, the resulting S-adenosylhomocysteine is not accumulated in the Golgi. SAM incorporation was also found to be inhibited by S-adenosylhomocysteine, whereas UDP-GalA, UDP-GlcA, and acetyl-CoA had no effect. DIDS, a compound that inhibits nucleotide sugar transporters, also had little effect upon SAM incorporation. Interestingly, the combination of UDP-GalA + acetyl-CoA or UDP-GlcA + acetyl-CoA produced a slight increase in the uptake of SAM. These results support the idea that a SAM transporter is required for HGA biosynthesis.     

The Rab GTPase Ypt1p and tethering factors couple protein sorting at the ER to vesicle targeting to the Golgi apparatus

GPI-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event can be reproduced in vitro. When extracts from a uso1 mutant were used, the sorting of GPI-anchored proteins from other secretory proteins was defective. Complementation with purified Uso1p restored sorting. The Rab GTPase Ypt1p and the tethering factors Sec34p and Sec35p, but not Bet3p, a member of the TRAPP complex, were also required for protein sorting upon ER exit. Therefore, the Ypt1p tethering complex couples protein sorting in the ER to vesicle targeting to the Golgi apparatus. Sorting of GPI-anchored proteins from other secretory proteins was also observed in vivo. The sorting defect observed in vitro with uso1 and ypt1 mutants was reproduced in vivo.     

Golgi manganese transport is required for rapamycin signaling in Saccharomyces cerevisiae

The Pmr1 Golgi Ca2+/Mn2+ ATPase negatively regulates target of rapamycin complex (TORC1) signaling, the rapamycin-sensitive TOR complex in Saccharomyces cerevisiae. Since pmr1 causes resistance to rapamycin and tor1 causes hypersensitivity, we looked for genetic interactions of pmr1 with tor1. Deletion of TOR1 restored two wild-type phenotypes. Loss of TOR1 restored the ability of the pmr1 strain to grow on media containing 2 mm MnCl2 and conferred wild type as well as the wild-type sensitivity to rapamycin. Mn2+ additions to media partially suppressed rapamycin resistance of wild type and pmr1 tor1, suggesting that Tor1 and Tor2 are regulated by manganese. We parsed the roles of Ca2+ and Mn2+ transport and the compartments in rapamycin response using separation-of-function mutants available for Pmr1. A strain containing the D53A mutant (Mn2+ transporting) of Pmr1 is rapamycin sensitive, but the Q783A mutant (Ca2+ transporting) strain is rapamycin resistant. Mn2+ transport into the Golgi lumen appears to be required for rapamycin sensitivity. Overexpression of Ca2+ pump SERCA1, Ca2+/H+ antiporter Vcx1, or a Mn2+ transporting mutant of Vcx1 (Vcx1-M1) failed to restore rapamycin sensitivity, and loss of Pmr1 but not other transporters of Ca2+ or Mn2+ results in rapamycin resistance. Overexpression of Ccc1, a Fe2+ and Mn2+ transporter that has been localized to Golgi and the vacuole, does restore rapamycin sensitivity to pmr1Delta. We conclude that Mn2+ in the Golgi inhibits TORC1 signaling.     

The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport

The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet-Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.     

Zebrafish fat-free is required for intestinal lipid absorption and Golgi apparatus structure

The zebrafish fat-free (ffr) mutation was identified in a physiological screen for genes that regulate lipid metabolism. ffr mutant larvae are morphologically indistinguishable from wild-type sibling larvae, but their absorption of fluorescent lipids is severely impaired. Through positional cloning, we have identified a causative mutation in a highly conserved and ubiquitously expressed gene within the ffr locus. The Ffr protein contains a Dor-1 like domain typical of oligomeric Golgi complex (COG) gene, cog8. Golgi complex ultrastructure is disrupted in the ffr digestive tract. Consistent with a possible role in COG-mediated Golgi function, wild-type Ffr-GFP and COG8-mRFP fusion proteins partially colocalize in zebrafish blastomeres. Enterocyte retention of an endosomal lipid marker in ffr larvae support the idea that altered vesicle trafficking contributes to the ffr mutant defect. These data indicate that ffr is required for both Golgi structure and vesicular trafficking, and ultimately lipid transport.     

Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro

Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that beta-COP, a component of non-clathrin- coated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of beta-COP specific antibodies and F(ab) fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that beta-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of beta-COP was found in a high molecular pool (> 1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rab1B could be quantitatively coprecipitated with this beta-COP containing complex and was also essential for function. We suggest that beta-COP functions in an early step during vesicle formation and that rab1B may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carriers.