The product of rab2, a small GTP binding protein, increases neuronal adhesion, and neurite growth in vitro.

The rab genes code for small GTP binding proteins that share with p21ras the ability to bind and hydrolyze GTP. They present significant sequence homologies with the products of YPT1 and SEC4, two small GTP binding proteins involved in the regulation of secretion in the yeast. Several rab genes are expressed in the developing and adult mouse brain. To test directly the possible involvement of these genes in neuronal differentiation, purified rab proteins produced in E. coli were introduced into neurons dissociated from E15 rat midbrain. The most striking effects were obtained with rab2 protein (rab2p). Compared with untreated cells, neurons loaded with rab2p presented an enhanced adhesion to the culture substratum. This phenomenon was visible 3 hr after seeding and was followed within 24 hr by a dramatic increase in neurite growth. Loading the same population of neurons with the products of four other rab genes either decreased neuronal adhesion and neurite growth or had no effect. These experiments suggest that the expression of rab2p plays an important role in neuronal differentiation.

Expression of the ras-related ralA, rho12 and rab genes in adult mouse tissues.

The expression of recently isolated mammalian ras-related ral, rho, and rab genes was examined in adult mouse tissues. Most of the genes studied were transcribed into two major messenger RNAs. The transcription of each gene appeared to be regulated in a complex manner with a tissue-specific modulation of the two transcripts. One member of the rab gene family, rab3, showed an RNA expression restricted to brain-tissues. Ral, rho, and three other rab genes had a more ubiquitous expression in murine tissues. However, the expression level of each gene showed a high degree of variation depending upon the organ. Among all the members of the enlarged ras gene family examined so far, the rab3 gene is the first example showing an expression restricted to a distinct organ.

Biochemical properties of the YPT-related rab1B protein. Comparison with rab1A.

We recently identified a novel rat cDNA: rab1B, closely related to the rab1A cDNA and to the yeast YPT1 gene. The rab1B cDNA encodes a 202 amino acid protein (22.1 kDa) that was produced in Escherichia coli under the control of the phi 10 promoter for the T7 RNA polymerase. The rab1B protein was purified in large amounts to near homogeneity in a simplified procedure. We studied the biochemical properties of rab1B and rab1A proteins. They both bind specifically GTP and GDP and possess intrinsic GTPase activities. The rab1B Lys21----Met mutant protein does not bind GTP, whereas the Ala65----Thr mutant has a reduced GTPase activity and is competent for autophosphorylation in the presence of GTP.

Transport between cis and medial Golgi cisternae requires the function of the Ras-related protein Rab6.

The small GTP-binding protein Rab6, a member of the Ras superfamily, is localized on the membranes of the Golgi apparatus and the trans Golgi network. Recent studies revealed that the Rab6 protein might be involved in the transit of proteins through the Golgi complex. In this report we demonstrate the essential function of the Rab6 protein in a distinct step of reconstituted Golgi transport. Polyclonal antibodies and Fab fragments directed against the C-terminal part of the Rab6 protein inhibit transport between the cis and the medial Golgi cisternae. Inhibition also occurred when a trans-dominant mutant form of the Rab6 protein (N126I) was added to the reconstituted transport. Furthermore, Rab6 antibodies inhibit uncoupled fusion of Golgi membranes. From these data we conclude that Rab6 is involved in a process related to membrane fusion at the cisternal membranes of the Golgi apparatus and therefore is needed for the consumption of Golgi-derived vesicles by their target membranes.

Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library.

Several oligonucleotide mixtures corresponding to a 6-amino acid sequence that is strictly conserved in all the ras and ras-related proteins (from various organisms) were tested for their ability to hybridize to 11 cloned members of the ras gene superfamily. Among these mixtures, a combination of two sets of partially complementary oligomers were able to hybridize to all the tested sequences. To identify members of the ras superfamily, we screened a rat brain cDNA library with these probes and isolated four genes, denoted rab1, -2, -3, and -4, encoding proteins homologous to the yeast YPT protein. Amino acid homology scores with YPT range from 75% for rab1 to 37% for rab4, whereas the homologies with p21 ras and other ras-related proteins are approximately equal to 30%, and homologous residues were clustered in the regions involved in GTP/GDP binding. Another striking similarity shared by the rab and the other ras-related proteins is the conservation of at least one cysteine residue near the carboxyl-terminal end involved in the membrane binding of the ras proteins. rab1 is a mammalian homolog of the yeast YPT gene, and the four rab genes constitute an additional branch of the ras gene superfamily that to our knowledge has not been described in higher eukaryotes.

The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion.

Seven cDNA clones corresponding to the rab1, rab2, rab3A, rab3B, rab4, rab5, and rab6 genes were isolated from a human pheochromocytoma cDNA library. They encode 23-25 kDa polypeptides which share approximately 30-50% homology and belong to the ras superfamily. The rab1, rab2, rab3A, and rab4 proteins are the human counterparts of the rat rab gene products that we have previously characterized. Comparison of the seven human rab proteins with the yeast YPT1 (YPT1p) and SEC4 (SEC4p) proteins reveals highly significant sequence similarities. H-rab1p shows 75% amino acid identity with YPT1p and may be therefore considered as its human counterpart. The other proteins share approximately 40% homology with YPT1p and SEC4p. The homology (approximately 30%) between these rab proteins and p21ras is restricted to the four conserved domains involved in the GTP/GDP binding. Human rab proteins were produced in Escherichia coli. Large amounts of rab proteins in soluble form can be extracted and purified without the use of detergents. All six proteins bind GTP and exhibit GTPase activities. A possible involvement of the rab proteins in secretion is discussed.

Developmental and regional expression of three new members of the ras-gene family in the mouse brain.

We have examined the expression in the mouse nervous system of three new members of the ras protooncogene family: rab1, rab2, and rab3. Each of these genes was transcribed into messenger RNAs with different molecular weights. These transcripts has specific developmental and regional patterns of expression. In particular, for the three genes, the ratio between the heavy and light mRNAs depended strongly on developmental stage and brain region. The use of pure neuronal and glial cultures revealed that the high molecular weight transcripts were enriched in neurons and that, in the case of rab2 and rab3, their expression increased with neuronal differentiation. These results are discussed considering the sequence identities between these genes and the yeast YPTI and sec-4 genes, which are known to be implicated in post-Golgi vesicular transport and cytoskeletal stabilization. We propose that the rab genes might be of importance in the regulation of these two processes within the developing and adult nervous system.

Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2.

Entry of a cell into mitosis induces a series of structural and functional changes including arrest of intracellular transport. Knowledge of how the mitotic cycle is driven progressed substantially with the identification of the p34cdc2 protein kinase as a subunit of maturation-promoting factor, the universal regulating component of the mitotic cycle. Activation of the kinase at the onset of mitosis is thought to trigger the important mitotic events by phosphorylating key proteins. Small guanine nucleotide-binding proteins have been implicated in regulating transport pathways. For instance, two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, control distinct stages of the secretory pathway in budding yeast. The GTP-binding proteins of the Rab family in rats and humans display strong homologies with Sec4p and Ypt1p, and might therefore also be involved in regulating intracellular transport. Indeed, distinct Rab proteins are located in the exocytotic and endocytotic compartments. Interruption of vesicular transport during mitosis might involve modification of these proteins. We now present biochemical evidence for a mitosis-specific p34cdc2 phosphorylation of Rab1Ap and Rab4p. By contrast, Rab2p and Rab6p are not phosphorylated. We also show that the distribution of Rab1Ap and Rab4p between cytosolic and membrane-bound forms is different in interphase and mitotic cells. This may provide a clue to the mechanism by which phosphorylation could affect membrane traffic during mitosis.

Chromosome assignment of four RAS-related RAB genes.

The human RAB genes share structural and biochemical properties with the RAS gene superfamily. The encoded RAB proteins show 38 to 75% amino acid identity with the yeast YPT1 and SEC4 gene products. We used four human RAB-cDNAs, RAB3B, RAB4, RAB5 and RAB6, to map the corresponding genes on human chromosomes. These genes were assigned to 1p32-p31, 1q42-q43, 3p24-p22 and 2q14-q21, respectively, by in situ hybridization.

Chromosome mapping of the human ras-related rab3A gene to 19p13.2.

The rab genes belong to one of the three main branches of the ras super family. The encoded rab proteins share 38 to 75% amino acid identity with the yeast YPT1 and SEC4 proteins. We used the human rab3A cDNA to map the corresponding gene on human chromosomes by chromosome sorting and in situ hybridization. Both techniques allowed the assignment of the rab3A gene to chromosome 19 with a regional localization on 19p13.2 obtained by in situ hybridization.

Palmaz stent placement in iliac and femoropopliteal arteries: primary and secondary patency in 310 patients with 2-4-year follow-up.

PURPOSE: To define the long-term outcome of stent placement in iliac and femoropopliteal arteries. MATERIALS AND METHODS: Three hundred ten patients received 418 balloon-expandable Palmaz stents. Two hundred thirty stents were implanted in iliac arteries of 184 patients, and 188 stents were implanted in femoropopliteal arteries in 126 patients. Restenosis rates were based on results of angiography performed 4-6 months after stent placement. Long-term patency rates were determined with duplex ultrasound. RESULTS: Immediate procedural success was achieved in 309 patients. Acute thrombosis ( < 24 hours) occurred in five patients, and immediate clinical success in 288. The 30-day mortality and morbidity rates were 0% and 8%, respectively. Angiography performed at 6 months in 299 patients revealed restenosis rates of 0.5% in iliac lesions, 11% in superficial femoral artery (SFA) lesions, and 20% in popliteal lesions. Survival analysis revealed 4-year primary patency rates of 86% +/- 4.1 for iliac artery lesions, 65% +/- 7.5 for SFA lesions, and 50% +/- 17.7 for popliteal artery lesions. Most restenotic lesions were successfully treated with repeat angioplasty. CONCLUSION: Implantation of Palmaz stents in iliac arteries allows long-term primary patency to be maintained in most patients.

Stent placement in the renal artery: three-year experience with the Palmaz stent.

PURPOSE: To evaluate the long-term results of stent placement in the renal arteries. PATIENTS AND METHODS: From January 1990 to August 1994, 59 hypertensive patients underwent 64 stent placement procedures. Indications were residual stenosis after percutaneous transluminal renal angioplasty in 42 patients, restenosis in 20 patients, and acute dissection in two patients. Follow-up (mean, 14 months) was obtained in 54 patients. Six-month restenosis rates were based on results of arteriography, and even more long-term patency rates were based on duplex ultrasound. RESULTS: Technical success was achieved in all patients. Major complications occurred in two patients. No minor or puncture-site complications were observed. The overall 6-month restenosis rate was 1.6% (2.9% for ostial lesions). Survival analysis with the Kaplan-Meier method showed primary and secondary patency rates of 92% +/- 3.6 and 98% +/- 1.9, respectively, at 1 year and 79% +/- 8.8 and 92% +/- 6.1, respectively, at 2 years. Seventy-six percent of hypertensive patients benefited from the procedure. However, renal function was not improved by stent placement. CONCLUSION: Stent placement in renal arteries is a useful adjunct to percutaneous transluminal angioplasty for atherosclerotic renal-artery stenoses.