The GTP-binding Sar1 protein is localized to the early compartment of the yeast secretory pathway

SAR1, the yeast gene which encodes a novel type of small GTP-binding protein, has been shown to be required for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To further the understanding of the function of its product, a lacZ-SAR1 hybrid gene was constructed and a polyclonal antibody was raised against the hybrid protein. This antibody specifically recognizes the SAR1 gene product (Sar1p) as a 23-kDa protein in the yeast cell lysate. We examined the subcellular localization of Sar1p using this antibody. In wild-type cells, Sar1p was predominantly recovered in a rapidly sedimenting membrane fraction that includes the ER. The soluble form of Sar1p was also detected when the protein was overproduced. Immunofluorescence microscopy with the anti-Sar1p antibody showed perinuclear staining that was exaggerated in the ER-accumulating sec18 mutant. Membrane association of Sar1p was shown to be very light. Sar1p was not extracted from the membrane by treatment with alkaline sodium carbonate, and only 1% deoxycholic acid solubilized Sar1p completely. From these results, we suggest that Sar1p is firmly located on the ER membrane where it regulates the ER-Golgi traffic.     

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.     

Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily.

In Saccharomyces cerevisiae, the GTP-binding Ypt1 protein (Ypt1p) is essential for endoplasmic reticulum-to-Golgi protein transport. By exploiting a GAL10-YPT1 fusion to regulate YPT1 expression, three multicopy suppressors, SLY2, SLY12, and SLY41, and a single-copy suppressor, SLY1-20, that allowed YPT1-independent growth were isolated. Wild-type Sly1p is hydrophilic, is essential for cell viability, and differs from Sly1-20p by a single amino acid. SLY2 and SLY12 encode proteins with hydrophobic tails similar to synaptobrevins, integral membrane proteins of synaptic vesicles in higher eucaryotes. Sly41p is hydrophobic and exhibits sequence similarities with the chloroplast phosphate translocator. SLY12 but not SLY41 is an essential gene. The SLY2 null mutant is cold and heat sensitive. The SLY gene products may comprise elements of the protein transport machinery.     

YOS9, the putative yeast homolog of a gene amplified in osteosarcomas,is involved in the endoplasmic reticulum (ER)-Golgi transport of GPI-anchored proteins

The OS-9 gene maps to a region (q13-15) of chromosome 12 that is highly amplified in human osteosarcomas and encodes a protein of unknown function. Here we have characterized a homolog designated as YOS9 (YDR057w) from Saccharomyces cerevisiae. The yeast protein (Yos9) is a membrane-associated glycoprotein that localizes to the endoplasmic reticulum (ER). YOS9 interacts genetically with genes involved in ER-Golgi transport, particularly SEC34, whose temperature-sensitive mutant is rescued by YOS9 overexpression. Interestingly, Yos9 appears to play a direct role in the transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus. Yos9 binds directly to Gas1 and Mkc7 and accelerates Gas1 transport and processing in cells overexpressing YOS9. Correspondingly, Gas1 processing is slowed in cells bearing a deletion in YOS9. No effect upon the transport and processing of non-GPI-anchored proteins (e.g. invertase and carboxypeptidase Y) was detected in cells either lacking or overexpressing Yos9. As Yos9 is not a component of the Emp24 complex, it may act as a novel escort factor for GPI-anchored proteins in ER-Golgi transport in yeast and possibly in mammals.     

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport.

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.     

Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator

Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.     

Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum.

Sec12p is a membrane glycoprotein required for the formation of a vesicular intermediate in protein transport from the endoplasmic reticulum to the Golgi apparatus in Saccharomyces cerevisiae. Comparison of the N-linked glycosylation of Sec12p, a Sec12p-invertase hybrid protein, and a derivative of Sec12p lacking 71 carboxy-terminal amino acids showed that Sec12p is a type II membrane protein. Analysis of two truncated forms of Sec12p and of a temperature-sensitive mutant indicated that the C-terminal domain of Sec12p is not essential for protein transport, whereas the integrity and membrane attachment of the cytoplasmic N-terminal domain are essential. Expression of a soluble cytoplasmic domain dramatically inhibited the growth of a sec12 temperature-sensitive strain by increasing the transport defect at a normally permissive temperature. This growth inhibition as well as the sec12 temperature-sensitive defect were suppressed by the overproduction of Sar1p, a small GTP-binding protein that participates in protein transport. Sar1p membrane association was enhanced by elevated levels of Sec12p. These results suggest that the cytoplasmic domain of Sec12p interacts with Sar1p and that the complex may function to promote vesicle formation.     

RPA, a class II ARFGAP protein, activates ARF1 and U5 and plays a role in root hair development in Arabidopsis

The polar growth of plant cells depends on the secretion of a large amount of membrane and cell wall materials at the growing tip to sustain rapid growth. Small GTP-binding proteins, such as Rho-related GTPases from plants and ADP-ribosylation factors (ARFs), have been shown to play important roles in polar growth via regulating intracellular membrane trafficking. To investigate the role of membrane trafficking in plant development, a Dissociation insertion line that disrupted a putative ARF GTPase-activating protein (ARFGAP) gene, AT2G35210, was identified in Arabidopsis (Arabidopsis thaliana). Phenotypic analysis showed that the mutant seedlings developed isotropically expanded, short, and branched root hairs. Pollen germination in vitro indicated that the pollen tube growth rate was slightly affected in the mutant. AT2G35210 is specifically expressed in roots, pollen grains, and pollen tubes; therefore, it is designated as ROOT AND POLLEN ARFGAP (RPA). RPA encodes a protein with an N-terminal ARFGAP domain. Subcellular localization experiments showed that RPA is localized at the Golgi complexes via its 79 C-terminal amino acids. We further showed that RPA possesses ARF GTPase-activating activity and specifically activates Arabidopsis ARF1 and ARF1-like protein U5 in vitro. Furthermore, RPA complemented Saccharomyces cerevisiae glo3Delta gcs1Delta double mutant, which suggested that RPA functions as an ARFGAP during vesicle transport between the Golgi and the endoplasmic reticulum. Together, we demonstrated that RPA plays a role in root hair and pollen tube growth, most likely through the regulation of Arabidopsis ARF1 and ARF1-like protein U5 activity.     

Calcium modulation of polyamine transport is lost in a putrescine-sensitive mutant of Neurospora crassa.

Putrescine transport in Neurospora is saturable and concentrative in dilute buffers, but in the growth medium putrescine simply equilibrates across the cell membrane. We describe a mutant, puu-1, that can concentrate putrescine from the growth medium because the polyamine transport system has lost its normal sensitivity to Ca2+. The wild type closely resembles the mutant if it is washed with citrate and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. The mutant phenotype also appears in the wild type after treatment with cycloheximide. The results suggest that putrescine uptake is normally regulated by an unstable Ca(2+)-binding protein that restricts polyamine uptake. This protein is evidently distinct from the polyamine-binding function for uptake, which is normal in mutant and in cycloheximide-treated wild type cells. The puu-1 mutation, stripping of Ca2+, and cycloheximide treatment all cause an impairment of amino acid transport, indicating that other membrane transport functions rely upon the product of the puu-1+ gene. Preliminary evidence suggests that the putrescine carrier is not the Ca(2+)-sensitive, low-affinity K(+)-transport system, but K+ efflux does accompany putrescine uptake.     

Cold-sensitive growth and decreased GTP-hydrolytic activity from substitution of Pro17 for Val in Era, an essential Escherichia coli GTPase

A substitution mutation of Pro17 by Val (P17V) was constructed in the guanine nucleotide binding domain of Era, an essential protein in Escherichia coli. The mutation is analogous to the oncogenic activating allele at position 12 in the GTP-binding domain of p21ras. The phenotype of this mutant was analysed in a strain which exclusively expressed the mutant protein (Era-V17) in null allele chromosomal background (era1: :kan). The strain was found to be cold-sensitive for growth. Mutant Era-V17 purified from the strain was cold-sensitive for GTP-hydrolytic activity, suggesting that the GTPase activity of Era is required for cell growth since the P17V mutation resulted in both cold-sensitive growth of cells and cold-labile GTPase activity of the purified protein.     

Temperature-sensitive lethal mutant of era, a G protein in Escherichia coli.

The era gene of Escherichia coli encodes a GTP-binding protein which has similarities to elongation factor Tu and the Saccharomyces cerevisiae RAS protein. To investigate its function, mutations affecting era were isolated. A mini-Tn10 insertion, which truncated 22 amino acids from the COOH end of Era, did not affect cell growth. By using this mini-Tn10 insert as a coselectable marker, a temperature-sensitive lethal era mutant was isolated by localized mutagenesis using P1 phage transduction. A single-base G to A change was found at position 23, causing a tyrosine residue to be substituted for the cysteine residue at position 8 (era-770), in addition to the COOH-terminal mini-Tn10 disruption. Both alterations were necessary for the temperature-sensitive phenotype. Purified Era-770 mutant protein exhibited reduced binding to GTP compared with that of the wild-type Era protein.     

Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport

Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.     

Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport

The yeast SNARE Ykt6p has been implicated in several trafficking steps, including vesicular transport from the endoplasmic reticulum (ER) to the Golgi, intra-Golgi transport, and homotypic vacuole fusion. The functional role of its mammalian homologue (Ykt6) has not been established. Using antibodies specific for mammalian Ykt6, it is revealed that it is found mainly in Golgi-enriched membranes. Three SNAREs, syntaxin 5, GS28, and Bet1, are specifically associated with Ykt6 as revealed by co-immunoprecipitation, suggesting that these four SNAREs form a SNARE complex. Double labeling of Ykt6 and the Golgi marker mannosidase II or the ER-Golgi recycling marker KDEL receptor suggests that Ykt6 is primarily associated with the Golgi apparatus. Unlike the KDEL receptor, Ykt6 does not cycle back to the peripheral ER exit sites. Antibodies against Ykt6 inhibit in vitro ER-Golgi transport of vesicular stomatitis virus envelope glycoprotein (VSVG) only when they are added before the EGTA-sensitive stage. ER-Golgi transport of VSVG in vitro is also inhibited by recombinant Ykt6. In the presence of antibodies against Ykt6, VSVG accumulates in peri-Golgi vesicular structures and is prevented from entering the mannosidase II compartment, suggesting that Ykt6 functions at a late stage in ER-Golgi transport. Golgi apparatus marked by mannosidase II is fragmented into vesicular structures in cells microinjected with Ykt6 antibodies. It is concluded that Ykt6 functions in a late step of ER-Golgi transport, and this role may be important for the integrity of the Golgi complex.     

Sec12p-dependent membrane binding of the small GTP-binding protein Sar1p promotes formation of transport vesicles from the ER

Sec12p is an integral membrane protein required in vivo and in vitro for the formation of transport vesicles generated from the ER. Vesicle budding and protein transport from ER membranes containing normal levels of Sec12p is inhibited in vitro by addition of microsomes isolated from a Sec12p-overproducing strain. Inhibition is attributable to titration of a limiting cytosolic protein. This limitation is overcome by addition of a highly enriched fraction of soluble Sar1p, a small GTP-binding protein, shown previously to be essential for protein transport from the ER and whose gene has been shown to interact genetically with sec12. Furthermore, Sar1p binding to isolated membranes is enhanced at elevated levels of Sec12p. Sar1p-Sec12p interaction may regulate the initiation of vesicle budding from the ER.     

Transport of acetate in mutants of Saccharomyces cerevisiae defective in monocarboxylate permeases

The strain Saccharomyces cerevisiae W303-1a, able to grow in a medium containing acetic acid as the sole carbon and energy source, was subjected to mutagenesis in order to obtain mutants deficient in monocarboxylate permeases. Two mutant clones exhibiting growth in ethanol, but unable to grow in a medium with acetic acid as the sole carbon and energy source, were isolated (mutants Ace12 and Ace8). In both mutants, the activity for the acetate carrier was strongly affected. The mutant Ace8 revealed not to be affected in the transport of lactate, while the mutant Ace12 did not display activity for that carrier. These results reinforced those previously found in the strain IGC 4072, where two distinct transport systems for monocarboxylates have been described, depending on the growth carbon source. It is tempting to postulate that the Ace8 mutant seems to be affected in the gene coding for an acetate permease. In contrast, the absence of activity for both monocarboxylate permeases in mutant Ace12 could be attributed to a mutation in a gene coding for a regulatory protein not detected before.     

Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era,an essential GTPase in Escherichia coli

Era is a small GTP-binding protein and essential for cell growth in Escherichia coli. It consists of two domains: N-terminal GTP-binding and C-terminal RNA-binding KH domains. It has been shown to bind to 16S rRNAs and 30S ribosomal subunits in vitro. Here, we report that a precursor of 16S rRNA accumulates in Era-depleted cells. The accumulation of the precursors is also seen in a cold-sensitive mutant, E200K, in which the mutation site is located in the C-terminal domain. The major precursor molecule accumulated seems to be 17S rRNA, containing extra sequences at both 5' and 3' ends of 16S rRNA. Moreover, the amounts of both 30S and 50S ribosomal subunits relative to the amount of 70S monosomes increase in Era-depleted and E200K mutant cells. The C-terminal KH domain has a high structural similarity to the RbfA protein, a cold shock protein that also specifically associates with 30S ribosomal subunits. RbfA is essential for cell growth at low temperature, and a precursor of 16S rRNA accumulates in an rbfA deletion strain. The 16S rRNA precursor seems to be identical in size to that accumulated in Era mutant cells. Surprisingly, the cold-sensitive cell growth of the rbfA deletion cells was partially suppressed by overproduction of the wild-type Era. The C-terminal domain alone was not able to suppress the cold-sensitive phenotype, whereas Era-dE, which has a 10-residue deletion in a putative effector region of the N-terminal domain, functioned as a more efficient suppressor than the wild-type Era. It was found that Era-dE suppressed defective 16S rRNA maturation, resuming a normal polysome profile to reduce highly accumulated free 30S and 50S subunits in the rbfA deletion cells. These results indicate that Era is involved in 16S rRNA maturation and ribosome assembly.     

Carbon tetrachloride suppresses ER-Golgi transport by inhibiting COPII vesicle formation on the ER membrane in the RLC-16 hepatocyte cell line

Carbon tetrachloride (CCl₄) causes hepatotoxicity in mammals, with its hepatocytic metabolism producing radicals that attack the intracellular membrane system and destabilize intracellular vesicle transport. Inhibition of intracellular transport causes lipid droplet retention and abnormal protein distribution. The intracellular transport of synthesized lipids and proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is performed by coat complex II (COPII) vesicle transport, but how CCl₄ inhibits COPII vesicle transport has not been elucidated. COPII vesicle formation on the ER membrane is initiated by the recruitment of Sar1 protein from the cytoplasm to the ER membrane, followed by that of the COPII coat constituent proteins (Sec23, Sec24, Sec13, and Sec31). In this study, we evaluated the effect of CCl₄ on COPII vesicle formation using the RLC-16 rat hepatocyte cell line. Our results showed that CCl₄ suppressed ER-Golgi transport in RLC-16 cells. Using a reconstituted system of rat liver tissue-derived cytoplasm and RLC-16 cell-derived ER membranes, CCl₄ treatment inhibited the recruitment of Sar1 and Sec13 from the cytosolic fraction to ER membranes. CCl₄-induced changes in the ER membrane accordingly inhibited the accumulation of COPII vesicle-coated constituent proteins on the ER membrane, as well as the formation of COPII vesicles, which suppressed lipid and protein transport between the ER and Golgi apparatus. Our data suggest that CCl₄ inhibits ER-Golgi intracellular transport by inhibiting COPII vesicle formation on the ER membrane in hepatocytes.     

Nitrogen regulation of urease synthesis in Saccharopolyspora erythraea ATCC 11365

Abstract Era is an essential GTP-binding protein of an unknown function in Escherichia coli . On the basis of its sequence similarities to other GTP-binding proteins such as E. coli EF-Tu, EF-G, IF2 and eukaryotic Ras proteins, it has been suggested that the Era function is activated by GTP binding, and that subsequent conversion of bound GTP to GDP by the intrinsic GTPase activity modulates its function. Two Era mutants, one dominant negative mutant (dE), which has a deletion mutation from Ala40 to Gly49, and the other non-functional mutant (T42A/T43A), which has two substitution mutations, Thr42 to Ala and Thr43 to Ala, were analyzed for their abilities of GTP-binding and GTPase activity. It was found that the dE mutant lost the GTP-binding ability, while it still retained the GTPase activity. On the other hand, the T42A/T43A mutant retained both the GTP-crosslinking and GTPase activities. However, the K _m values for GTPase activity increased 5-and 12-fold for dE and T42A/T43A mutants, respectively. These results indicate that both the GTP-binding and GTPase activities are important for the Era function.     

Rate of protein transport in thyroid follicle cells of normal, thyroxine-treated and TSH-injected rats

The transport of labeled protein in thyroid follicles was studied with quantitative electron microscopic autoradiography in normal and T4-treated rats (2d) injected with 3H-leucine 1 to 6 h before perfusion fixation. During this time interval the total amount of labeled protein in either group was unchanged, although T4-treatment caused a reduction by about 30% of the amount of 3H-leucine incorporated into protein. The autoradiographic data were corrected for the effect of scatter of radioactivity. The relative amounts of labeled, exportable protein in the compartments Er-Golgi and exocytotic vesicles were then estimated. The half-lives of labeled, exportable protein in these compartments were calculated with non-linear regression analysis. In normal rats the half-life of labeled, exportable protein in ER-Golgi was 28 min and in the exocytotic vesicles 18 min. Inhibition of TSH-secretion by injection of thyroxine decreased the rate of protein transport through the follicle cell and increased the half-lives to 63 min (ER-Golgi) and 62 min (exocytotic vesicles). TSH given to thyroxine-treated rats 20 min or 1.5 h before fixation reduced the half-lives of labeled, exportable protein in ER-Golgi to 25 to 33 min and in exocytotic vesicles to 9 min. The findings indicate that TSH regulates the rate of intracellular protein transport in rat thyroid follicle cells at the exocytotic step as well as at an earlier step in the pathway of intracellular protein transport. The mechanism and exact location of the latter TSH regulated step is at present unknown.     

The arabidopsis cell plate-associated dynamin-like protein, ADL1Ap, is required for multiple stages of plant growth and development

Dynamin and dynamin-like proteins are GTP-binding proteins involved in vesicle trafficking. In soybean, a 68-kD dynamin-like protein called phragmoplastin has been shown to be associated with the cell plate in dividing cells (Gu and Verma, 1996). Five ADL1 genes encoding dynamin-like proteins related to phragmoplastin have been identified in the completed Arabidopsis genome. Here we report that ADL1Ap is associated with punctate subcellular structures and with the cell plate in dividing cells. To assess the function of ADL1Ap we utilized a reverse genetic approach to isolate three separate Arabidopsis mutant lines containing T-DNA insertions in ADL1A. Homozygous adl1A seeds were shriveled and mutant seedlings arrested soon after germination, producing only two leaf primordia and severely stunted roots. Immunoblotting revealed that ADL1Ap expression was not detectable in the mutants. Despite the loss of ADL1Ap, the mutants did not display any defects in cytokinesis, and growth of the mutant seedlings could be rescued in tissue culture by the addition of sucrose. Although these sucrose-rescued plants displayed normal vegetative growth and flowered, they set very few seeds. Thus, ADL1Ap is critical for several stages of plant development, including embryogenesis, seedling development, and reproduction. We discuss the putative role of ADL1Ap in vesicular trafficking, cytokinesis, and other aspects of plant growth.