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© 1996 Oxford University Press 2005-2010

Footnote

Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9

Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9 Toshiharu Yasugi and Peter M. Howley*

Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston , MA 02115, USA

Received March 18, 1996; Accepted April 12, 1996 GenBank accession no. X96427

ABSTRACT

Ubiquitin conjugating enzymes (UBCs) are a family of proteins directly involved in ubiquitination of proteins. Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation. The ubc9 gene of the yeast Saccharomyces cerevisiae (Sc ubc9 ) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins. We have identified a human homolog of Sc ubc9 (termed h ubc9 ) using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein. The h ubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5 + gene product of Schizosaccharomyces pombe . Genetic complementation experiments in a S.cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression.

INTRODUCTION

The ubiquitin-dependent proteolytic system is a major pathway for the selective degradation of proteins in eukaryotes. This proteolytic system plays an important role in controlling the levels of key enzymes and regulatory proteins and in preventing the accumulation of abnormal proteins in cells ( 1 - 4 ). Ubiquitination of proteins is accomplished by an ATP-dependent multi-step pathway that is initiated by the activation of ubiquitin by ubiquitin activating enzymes, following by substrate-specific conjugation by ubiquitin conjugating enzymes (UBCs) and ubiquitin-protein ligases ( 1 - 4 ). The UBCs participate in the transfer of ubiquitin to a target protein either directly or through the participation of a ubiquitin-protein ligase, such as Ubr1 ( 5 ), E3[alpha] ( 2 ), E3[beta] ( 2 ) and E6-AP ( 6 ). The covalent linkage of ubiquitin to substrate proteins serves to signal their degradation by the 26S proteasome ( 7 ).

Members of the UBC proteins contain a highly conserved catalytic site. Previous studies in Saccharomyces cerevisiae have identified at least 10 different UBCs that are involved in various cellular processes, such as DNA repair, sporulation, cell cycle progression, heat shock resistance and peroxisome biogenesis ( 1 - 3 ). Among these S.cerevisiae UBCs, the yeast enzymes UBC3/CDC34 and UBC9 are the only ones that have been shown individually to be essential for cell viability ( 3 ). Repression of ScUBC9 synthesis prevents cell cycle progression at the G2 or early M phase, causing the accumulation of large budded cells with a single nucleus, a short spindle and replicated DNA ( 8 ). Furthermore, the accumulation of both CLB5, an S phase cyclin and CLB2, an M phase cyclin, has been observed in mutants of ScUBC9 ( 8 ). The Schizosaccharomyces pombe hus5 + gene product, a structural homolog of ScUBC9, is also required for normal mitosis ( 9 ).

We have now isolated and characterized a human UBC, termed hUBC9, because of the high degree of similarity of the predicted amino acid sequence to ScUBC9. The h ubc9 gene encodes a transcript of 1.3 kb and the h ubc9 open reading frame encodes a protein of 158 amino acids with a predicted molecular size of 18 kDa. The expression of hUBC9 protein can support the growth of ubc9 temperature sensitive mutants at non-permissive temperatures, indicating that hUBC9 is a functional homolog of ScUBC9.

MATERIALS AND METHODS

Isolation of h ubc9 cDNA

A yeast two hybrid screen was performed according to previously developed systems ( 10 - 12 ) as modified by Dr Marc Vidal ( 13 ). The bait plasmid was constructed by inserting the full-length E1 gene of human papillomavirus type 16 (HPV16) in-frame with the GAL4 DNA binding domain (amino acids 1-147) in the pPC97 vector (pPC97-16E1). The pPC97 and pPC86 vectors, an activated human T cell cDNA library cloned in the pPC86 vector and the yeast host strain MaV103 (MAT a ura3-52 leu2-3,112 trp1-901 his3 [Delta] 200 ade2-101 gal4 [Delta] gal80 [Delta] GAL1 :: LacZ GAL1 :: HIS3@lys2 SPAL10 :: URA3 ) were kindly provided by Drs Marc Vidal and Joshua La Baer (Massachusetts General Hospital, Charlestown, MA) ( 13 ). Selection was based on the presence of the HIS3 gene cloned downstream of GAL4 DNA binding sites. Increased HIS3 expression results as a consequence of an interacting clone. Since HIS3 expression renders yeast resistant to 3-aminotriazole (3AT), different concentrations of 3AT can be used to select interacting clones. The selection of interaction-positive clones was performed on plates containing 75 mM 3AT to reduce the background growth of the yeast transformants harboring pPC97-16E1. pPC86-derived prey plasmids containing cDNA were rescued from positive clones by transformation of competent DH5[alpha] bacteria with total yeast DNA. The sequence of both strands of the h ubc9 cDNA insert was determined with appropriate synthetic oligonucleotide primers by dideoxynucleotide sequencing using Sequenase 2.0 (United States Biochemical).

Plasmid construction

The Sc ubc9 gene was amplified by polymerase chain reaction (PCR) using appropriate synthetic oligonucleotides and cloned into yeast centromeric plasmid pRS316 (Stratagene), yielding pRS316-ScUBC9. The pRS316 vector contains the URA3 selectable marker. The yeast expression plasmid pTY316 was constructed by inserting a pPC86-derived Kpn I- Hin dIII fragment of the highly expressed S.cerevisiae ADC1 promoter into the pRS316 vector (Stratagene). A Not I restriction fragment of the pPC86 library plasmid containing h ubc9 cDNA was subcloned downstream of the ADC1 promoter in pTY316 (pTY316-hUBC9wt). The substitution mutant of h ubc9 (Cys -> Ser at codon 93, TGC -> AGC) was generated using PCR-mediated, site-directed mutagenesis ( 14 ). This h ubc9 mutant (h ubc9mt ) was also cloned downstream of the ADC1 promoter in pTY316 (pTY316-hUBC9mt). To obtain yeast expression plasmids for epitope-tagged hUBC9wt and hUBC9mt, fragments containing the full coding region of h ubc9 cDNA along with the influenza virus hemagglutinin 1 epitope (HA) sequence (YPYDVPDYA) at the 3'-end were produced with appropriate synthetic oligonucleotides using PCR. These fragments were cloned into the pCMV 4 vector ( 15 ) at a Bgl II site, yielding pCMV-hUBC9wtHA and pCMV-hUBC9mtHA. The Eco RI restriction fragments from these pCMV 4 -derived plasmids, containing HA-tagged h ubc9 cDNA and the human growth hormone terminator sequence, were cloned downstream of the ADC1 promoter in pTY316, yielding plasmids pTY316-hUBC9wtHA and pTY316-hUBC9mtHA.

Saccharomyces cerevisiae strains

The S.cerevisiae UBC9 temperature sensitive mutant ( ubc9ts ) strain YWO102 (MAT[alpha], ubc9- [Delta] 1 :: TRP1 , LEU2 :: ubc9-1 ) ( 9 ) was kindly provided by Dr Stefan Jentsch. The S.cerevisiae UBC3/CDC34 temperature sensitive mutant ( ubc3ts ) strain KY203 (MAT a , ura3-52 , leu2- [Delta] 2 , bas1-2 , bas2-2 , gcn4- [Delta] 1 , [alpha][delta][epsilon] 8-GCN4 , cdc34-2 ) ( 16 ) was kindly provided by Dr Daniel Kornitzer. Standard genetic techniques for S.cerevisiae were used ( 17 ).

Northern blot analysis

The Not I restriction fragment of cDNA including the protein coding sequence plus the 3'-UTR of h ubc9 and the human [beta]-actin cDNA fragment were radiolabeled with [[alpha]- 32 P]dCTP. The radiolabeled probes were hybridized to a multi-tissue Northern (MTN) blot (Clontech). The blot was hybridized at 60oC and washed at 65oC in 0.1* SSC, 0.1% SDS according to the manufacturer's instructions. The blot was then exposed to X-ray film overnight with double intensity screens at -70oC.

Western blot analysis


Figure 1 . The nucleotide sequence of the h ubc9 cDNA. The start codon ATG at position +1 and the stop codon TAA at position +475 are double underlined. A presumed polyadenylation signal is single underlined. Amino acids are indicated in the single letter code.Total yeast cell lysates were prepared by directly adding SDS-PAGE sample buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) to cell pellets. The samples were electrophoresed by 15% SDS-PAGE and the proteins were transferred to Immobilon-P membranes (Millipore Corp.). Western blotting and chemiluminescent detection were performed following the manufacturer's protocol (ECL Western detection kit; Amersham).

RESULTS

Isolation and primary sequence of hUBC9

In studies designed to examine the functions of HPV16 E1, a protein essential for the initiation of viral DNA replication, we used the yeast two hybrid system to identify cDNA clones that encode proteins that can interact with the HPV16 E1 protein. The prey library was introduced into the yeast reporter strain MaV103 containing pPC97-16E1. Approximately 1.0 * 10 7 primary library transformants were plated out onto histidine dropout plates containing 75 mM 3AT. Five independent clones containing an identical ~1300 bp cDNA were found among a total of 16 positive clones from this two hybrid screen.


Figure 2 . ( A ) Comparison of the amino acid sequence of hUBC9 with other human UBCs: hUBC2 (19), hUBC4 (20), UBCH5 (21), UBCH6 (22), UBCH7 (22), hUBC8 (23) and E2-EPF (24). Identical amino acids are indicated in bold. The active site cysteine residue is marked with a star. The amino acid numbering shown denotes residues of the hUBC9 protein. ( B ) Comparison of amino acid sequence between hUBC9 and ScUBC9 (8). Identical amino acids are shown in white letters on a black background and the related amino acids are boxed in gray. The regions especially conserved between these two proteins are underlined. The active site cysteine residue is marked with a star. The amino acid numbering shown denotes residues of the hUBC9 protein.


pPC86 plasmids containing the cDNA fragments were rescued from positive yeast clones and the primary sequence of the cDNA was determined. The primary sequence shown in Figure 1 contains an open reading frame (ORF) that encodes 158 amino acids with a total predicted molecular mass of ~18 kDa. The deduced amino acid sequence encoded by this ORF has similarities with the family of known human UBCs (19-38% identity) (Fig. 2 A) and a putative conserved active site cysteine residue at codon 93 (Cys93), suggesting that the isolated cDNA encodes a new member of human UBCs. This new human UBC was termed hUBC9 because amino acid sequence comparison demonstrates that hUBC9 is highly similar to ScUBC9 (56% identity, 72% similarity) (Fig. 2 B). The comparison reveals two domains that are especially well conserved between hUBC9 and ScUBC9. One of these domains contains amino acids 8-24 (82% identity) and the other contains amino acids 83-108 (88% identity). These conserved regions may be important for their function. There is also significant similarity between hUBC9 and the Schizosaccharomyces pombe hus5 + gene product (data not shown). While this manuscript was in preparation, a cDNA sequence identical to that of the h ubc9 gene reported here was submitted to GenBank (M.Tachibana, unpublished results, accession no. U45328).

Northern blot analysis

The tissue distribution of h ubc9 expression was determined by Northern blot analysis using h ubc9 cDNA as a probe to hybridize polyadenylated RNAs (~2 [mu]g/lane; Clonetech) derived from a variety of human tissues. As shown in Figure 3 the h ubc9 probe detected a transcript of 1.3 kb in each of the human tissues examined, indicating that h ubc9 is ubiquitously expressed. Expression of this 1.3 kb polyadenylated RNA was also detected in the immortalized human keratinocyte cell line HaCaT (data not shown).


Figure 3 . Expression of h ubc9 mRNA in human tissues. Northern blot analysis of polyadenylated RNAs (~2 [mu]g/lane; Clonetech) derived from the indicated tissues probed with a h ubc9 fragment as described in Materials and Methods. The bottom panel shows rehybridization of the same blot to a [beta]-actin probe.

Functional complementation of a ubc9 mutation of S.cerevisiae by h ubc9

The high degree of amino acid identity between hUBC9 and ScUBC9 suggested that the human gene might function similarly to Sc ubc9 . To examine this possibility, the h ubc9 gene was tested for its ability to complement the cell cycle progression defects of a ubc9 temperature sensitive (ts) mutant in S.cerevisiae . Cells harboring the ubc9ts mutation grow normally at 25oC and fail to grow at the non-permissive temperature, >35oC ( 8 ). For these experiments, epitope-tagged versions of the wild-type h ubc9 gene, as well as a mutant h ubc9 gene in which codon 93 was changed to encode serine instead of cysteine were cloned into a yeast expression plasmid. These plasmids, designated pTY316-hUBC9wtHA and pTY316-hUBC9mtHA, were introduced into the S.cerevisiae ubc9ts mutant strain YWO102 by transformation. Plasmids pRS316-ScUBC9 and pTY316 were also introduced into YWO102 as positive and negative controls, respectively. Each of the transformants was streaked on plates containing selective minimal glucose medium and their growth was assessed at 25 and 37oC. Growth of YWO102 transformants was observed at 37oC for the transformants containing the positive control pRS316-ScUBC9, for transformants containing pTY316-hUBC9wtHA (Fig. 4 upper), as well as for transformants harboring the plasmid pTY316-hUBC9wt expressing a non-tagged version of the h ubc9 gene (data not shown). No growth was observed at 37oC for transformants harboring pTY316-hUBC9mt (data not shown) or transformants harboring the plasmid pTY316-hUBC9mtHA (Fig. 4 upper). These results indicate that hUBC9 can complement the function of ScUBC9 and that the HA epitope tagged to the C-terminus does not affect hUBC9 function. The expression of hUBCBC9mt (C93S) could not complement the function of ScUBC9, indicating that the conserved cysteine of hUBC9 at codon 93 is required for the function of hUBC9 as a ubiquitin conjugating enzyme. Equivalent expression of both HA-tagged wild-type hUBC9 and the C93S mutant hUBC9 in YWO102 was documented by Western blot analysis using a HA-specific monoclonal antibody 12CA5 (Fig. 5 ).


Figure 4 . Specific functional complementation of ScUBC9 by the introduction of hUBC9. Transformants of YWO102 or KY203 harboring the indicated plasmids were streaked on selective plates and were incubated at 25 and 37oC. Photographs were taken after 4 days incubation.


Figure 5 . Expression of the HA-tagged wild-type and the C93S mutant hUBC9 proteins in yeast. Transformants of YWO102 or KY203 harboring the indicated plasmids were harvested from 4 ml liquid culture at mid log phase (optical density 600 nm ~0.7). Monoclonal antibody 12CA5 was used to detect HA-tagged proteins.

To examine whether the ability of hUBC9 to promote the growth of YWO102 was mediated by specific complementation of ScUBC9 function, hUBC9 was also tested for its ability to complement the ubc3ts mutant strain KY203. KY203 was transformed with the plasmids pTY316-hUBC9wtHA and pTY316-hUBC9mtHA. There was no growth of the transformants at 37oC (Fig. 4 lower), despite adequate expression of both wild-type and mutant forms of hUBC9 in KY203 (Fig. 5 ), indicating that hUBC9 could not complement the function of S.cerevisiae UBC3/CDC34. These results demonstrate that growth of the YWO102 transformants was mediated by specific complementation of the ScUBC9 function by hUBC9.

DISCUSSION

Here, we report the cDNA cloning and characterization of a human ubiquitin conjugating enzyme, hUBC9, that is a structural and functional homolog of ScUBC9. The high degree of structural and functional conservation between hUBC9 and ScUBC9 implies that both human and yeast proteins may interact with specific cellular factors that are also well conserved between human and yeast. Overall there is >50% amino acid identity between hUBC9 and ScUBC9. There are two regions that are particularly well conserved, one flanking Cys93 and the other consisting of amino acids Arg8-Phe24. The region flanking Cys93 is conserved among the various human UBCs, suggesting that this region may be involved in interactions with ubiquitin activating enzyme, because the cysteine residue conserved among the UBCs participates in the thioester linkage with ubiquitin, which is transferred directly to UBCs from the ubiquitin activating enzyme. The other conserved region, including amino acids Arg8-Phe24, might be involved in interactions with a specific ubiquitin-protein ligase or directly with the specific cellular targets, because the high degree of conservation of amino acids in this region does not extend to other human UBCs (Fig. 2 A). There are several studies that suggest the functional importance of S.cerevisiae UBCs N-terminal amino acid sequences. In the case of UBC8, the first 12 amino acids of the N-terminus are important for ubiquitination of histones in vitro ( 23 ). Watkins et al . have reported that the first nine amino acids of RAD6 (UBC2) are required for its physical interaction with yeast Ubr1 and that deletion of this region affects RAD6 function in sporulation and DNA repair ( 25 ). Mutational analysis of UBC9 will be required to clarify the role of these conserved domains.

Loss of ScUBC9 function in yeast cells results in the accumulation of the B-type cyclins CLB5 and CLB2 ( 8 ). The ability of hUBC9 to drive cell cycle progression in S.cerevisiae with a ubc9ts mutation raises the intriguing possibility that hUBC9 may also be involved in the ubiquitination of specific cyclins and play an important role in cell cycle progression in human cells. Identification of the specific cellular targets of hUBC9 may help to elucidate its exact role in cell cycle progression.

Although h ubc9 cDNA was identified through a two hybrid screen using HPV16 E1 protein, the significance of the interaction between HPV16 E1 and hUBC9 has not yet been determined. The E1 proteins of the papillomaviruses are essential for the initiation of viral DNA replication and have been shown to have ATP binding, ATPase and helicase activities ( 15 , 26 ). The levels of the E1 protein are believed to be quite low in infected cells and it is not known whether E1 levels are regulated in a cell cycle-dependent manner. Nonetheless, the interaction of hUBC9 and HPV16 E1 in yeast raises the possibility that E1 may be a target of the ubiquitin-dependent proteolytic pathway and that the E1 protein levels in HPV-infected cells might be regulated by a pathway governed by hUBC9. Alternatively, E1 might somehow modulate the activity of hUBC9 for the benefit of efficient viral DNA replication and might therefore influence cell cycle progression of mammalian cells. These hypotheses will be tested by further experiments trying to determine the physiological significance of the E1-hUBC9 interaction.

ACKNOWLEDGEMENTS

We thank Drs Marc Vidal and Joshua La Baer for the vectors, the yeast strain of the two-hybrid system and the human T cell library. We also thank Drs Stefan Jentsch and Daniel Kornitzer for providing yeast strains. We are grateful to Charles Ro for DNA sequencing and to Drs John D.Benson, Sushant Kumar and Hiroyuki Sakai for helpful discussions and comments on the manuscript. This work was supported in part by a Sponsored Research Agreement to Harvard University from the Terumo Corporation of Japan.

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A. V. Ivanova, S. V. Ivanov, A. Danilkovitch-Miagkova, and M. I. Lerman
Regulation of STRA13 by the von Hippel-Lindau Tumor Suppressor Protein, Hypoxia, and the UBC9/Ubiquitin Proteasome Degradation Pathway
J. Biol. Chem., April 27, 2001; 276(18): 15306 - 15315.
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 Proc. Natl. Acad. Sci. USAHome page
A. L. Kurtzman and N. Schechter
Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification
PNAS, May 8, 2001; 98(10): 5602 - 5607.
[Abstract] [Full Text] [PDF]

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