1 Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, George Washington University, School of Medicine and Health Sciences, 111 Michigan Avenue NW, Washington, DC 20010, USA
2 Department of Pediatrics, George Washington University, School of Medicine and Health Sciences, 111 Michigan Avenue NW, Washington, DC 20010, USA
Correspondence
Anamaris M. Colberg-Poley
acolberg-poley{at}cnmcresearch.org
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ABSTRACT |
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MAIN TEXT |
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The UL37x1 (pUL37x1), UL37 glycosylated (gpUL37) and UL37 medium (pUL37M) proteins are products of alternatively processed RNAs (Fig. 1a) (Adair et al., 2003
; Al-Barazi & Colberg-Poley, 1996
; Goldmacher et al., 1999
; Kouzarides et al., 1988
; Mavinakere & Colberg-Poley, 2004
; Tenney & Colberg-Poley, 1991
). The UL37x1 ORF, common to these UL37 proteins, contains two anti-apoptotic domains (Hayajneh et al., 2001a
; McCormick et al., 2003
). The first, spanning aa 534, includes a prominent hydrophobic leader and downstream basic residues. This bipartite signal targets UL37 proteins into the endoplasmic reticulum (ER) and to mitochondria (Mavinakere & Colberg-Poley, 2004
) where they prevent cytochrome c release following apoptotic signals (Goldmacher et al., 1999
). The second UL37 anti-apoptotic domain (aa 118147) is required for the association of pUL37x1 with Bax, resulting in the tight association of Bax with the mitochondrial outer membrane and inhibition of its pro-apoptotic function (Poncet et al., 2004
).
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ER targeting signals of secretory proteins are defined by a hydrophobic segment of the nascent polypeptide, which is often removed by cleavage following ER translocation (Johnson & van Waes, 1999). According to the (3, 1) rule, the UL37x1 hydrophobic leader is predicted to be non-cleavable because of the presence of two large residues (Phe and Tyr) in the critical 3 and 1 positions (Kouzarides et al., 1988
; Nielsen et al., 1997
). pUL37x1, the predominant UL37 protein during permissive HCMV infection, is ER-translocated and mitochondrially imported (Goldmacher et al., 1999
; Mavinakere & Colberg-Poley, 2004
). gpUL37 is produced at exceedingly low amounts in HCMV-infected HFFs and traffics through the ER, where it is N-glycosylated, and then to the Golgi apparatus (Al-Barazi & Colberg-Poley, 1996
). We have previously detected gpUL37 similarly trafficking into the secretory apparatus and into mitochondria in transiently transfected HFFs and HeLa cells (Colberg-Poley et al., 2000
).
In this communication, we show a unique pattern of differential trafficking coupled with post-translational processing of HCMV gpUL37. We observed a previously unreported internal consensus signal peptidase cleavage site in UL37x3 (aa 193/194) using SIGNAL IP 1.1 (Nielsen et al., 1997) within a compact hydrophobic domain identified using HMMTOP 1.1 (Tusnády & Simon, 1998
) (Fig. 1a
). Therefore, we anticipated that gpUL37 might be internally cleaved. This consensus cleavage site is well conserved in the chimpanzee cytomegalovirus (CCMV) UL37 ORF (Davison et al., 2003
) and is reminiscent of the internal cleavage site of influenza CM2 glycoprotein (Perkosz & Lamb, 1998
).
To gain further insight into its processing and trafficking, HeLa cells expressing gpUL37Flag were fractionated as described previously (Mavinakere & Colberg-Poley, 2004). To determine whether gpUL37 is internally cleaved, we used antibodies (Abs) against the N (Ab1064) (Fig. 1b
) and C (anti-Flag) termini (Fig. 1c
) of gpUL37Flag. Ab1064, which recognizes UL37x1 aa 2740, reacted with
(
27 kDa) in banded mitochondria (Fig. 1b
, lane 1) and intermediate as well as ER fractions (Fig. 1b
, lanes 24). The molecular mass of
corresponded well with cleavage at UL37x3 aa 193/194. A minor, non-specific band (
28 kDa) was weakly detected by Ab1064 in ER and total protein (Fig. 1b
, lanes 8 and 9) but not in purified mitochondria (Fig. 1b
, lane 7) of untransfected cells. Anti-Flag Ab detected cleaved gpUL37COOHFlag (
70 kDa), which was larger than predicted, in purified ER (Fig. 1c
, lane 4) and in intermediate fractions (Fig. 1c
, lanes 2 and 3). Surprisingly, neither Ab1064 nor anti-Flag Ab readily detected the uncleaved UL37 precursor, with predicted masses of
60 kDa (unglycosylated) or
98 kDa (N-glycosylated) in either ER or intermediate fractions. This latter finding suggests that proteolytic cleavage of the UL37 precursor is rapid and efficient and that the Western blot analyses were not sufficiently sensitive to detect the short-lived, intermediate species of gpUL37 protein processing, as it was weakly observed in radiolabelled immunoprecipitates from tunicamycin-treated, HCMV-infected HFFs (Al-Barazi & Colberg-Poley, 1996
). Reactivity of purified and intermediate fractions with Abs against ER (anti-dolichyl phosphate mannose synthase 1, anti-DPM1) and mitochondrial (anti-glucose regulated protein 75, anti-GRP75) markers indicated the enrichment of the corresponding markers (Fig. 1d and e
, lanes 19). In addition to the DPM1 subunit (
30 kDa), a large band (>
100 kDa) from the ER of gpUL37-expressing cells reacted with anti-DPM1 Ab (Fig. 1d
, lane 4). Because of its size, this species likely contains non-denatured complexes containing DPM1. Taken together, these results suggested that gpUL37 is proteolytically cleaved in the ER and that its products are stable in transfected cells. Moreover,
and UL37COOH dissociate and traffic to distinct subcellular compartments.
As UL37COOH was larger than predicted following proteolytic cleavage and contains multiple N-glycosylation sites, we examined whether N-glycosylation accounted for its increased molecular mass. Purified mitochondrial and ER fractions from the transfected HeLa cells expressing gpUL37Flag and treated with tunicamycin, an inhibitor of N-glycosylation, or untreated were examined by reactivity with Ab1064, anti-Flag, anti-DPM1 and anti-GRP75 Abs (Fig. 2). Ab1064 detected
in the mitochondria of untreated transfected cells but in trace amounts in tunicamycin-treated cells (Fig. 2
, top panel, lanes 3 and 4), suggesting that
from unglycosylated UL37 protein is unstable or that its epitope reactive with Ab1064 is unavailable.
migrated more slowly than pUL37x1 purified from the ER of HCMV-infected HFFs (Fig. 2
, top panel, lanes 3 and 9), consistent with pUL37 cleavage at the consensus peptidase site within UL37x3, approximately 30 residues downstream of the termination site of the UL37x1 ORF. UL37COOH from the ER of tunicamycin-treated cells detected by anti-Flag Ab was reduced in molecular mass (
3233 kDa) compared with that from untreated cells (
70 kDa; Fig. 2
, second panel, lanes 7 and 8). The molecular mass of the unglycosylated UL37COOH corresponded well with cleavage at the predicted site. Purified mitochondria (Fig. 2
, third panel, lanes 14) reacted well with anti-GRP75 but not detectably with DPM1 Ab (Fig. 2
, bottom panel, lanes 14). Conversely, purified ER fractions (Fig. 2
, bottom panel, lanes 59) reacted primarily with anti-DPM1 Ab and weakly with anti-GRP75 Ab (Fig. 2
, third panel, lanes 59). These results showed that UL37COOH is N-glycosylated in the ER and that pUL37 proteolytic cleavage does not require prior N-glycosylation. The abundance of UL37COOH carrying the N-glycosylation domain was minimally affected by tunicamycin treatment. In contrast, the abundance of
was dramatically reduced in tunicamycin-treated cells. Probable explanations for this unexpected finding include the possibilities that, in absence of N-glycosylation of the full-length UL37 precursor,
is misfolded and targeted for degradation or that its epitope for Ab1064 is masked by misfolding or proteinprotein interactions and hence unavailable.
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There are few examples of proteolytic cleavage of internal signal sequences except for proteins that span membranes more than once (Perkosz & Lamb, 1998). The capsid precursors of rubella virus (Oker-Blom et al., 1990
) and hepatitis C virus (Grakoui et al., 1993
) have internal signal peptidase cleavage sites. Cleavage of the influenza C virus gp42 internal signal peptidase site generates p31 and the glycosylated integral membrane protein CM2 (Perkosz & Lamb, 1998
). In contrast to our findings, one of the gp42 fragments, the p31 protein, could not be detected in infected cells. Both UL37 cleavage products (
and UL37COOH) were detected in transfected cells indicating that the N-terminal fragment (
) is not degraded but rather is imported into mitochondria.
Alternative processing of HCMV UL37 transcripts generates at least 11 different unspliced and spliced transcripts (Adair et al., 2003; Goldmacher et al., 1999
; Kouzarides et al., 1988
; Su et al., 2003
). Alternatively spliced UL37 RNAs are predicted to encode at least three additional UL37 isoforms. The UL37di and dii ORFs include the UL37x3 consensus signal peptidase cleavage site whereas UL37s (sdi and sdii) ORF does not. Thus, in addition to diversity resulting from alternative UL37 RNA processing, other UL37 isoforms are generated by pUL37 precursor cleavage in the ER as demonstrated by these studies. Because of their distinct domains, the UL37 isoforms will likely differ in functions as well.
The HCMV glycoprotein gB is cleaved during its trafficking through the cellular secretory apparatus (Britt & Auger, 1986; Singh & Compton, 2000
). However, gB is cleaved by furin as it traffics through the Golgi apparatus and forms inter-fragment dimers (Vey et al., 1995
). In contrast to gpUL37, the HCMV gB cleavage fragments are covalently linked by disulfide bridges, traffic jointly through the secretory apparatus and are both incorporated into the viral envelope (Britt & Auger, 1986
; Lopper & Compton, 2002
). gpUL37 fragments dissociate and traffic differentially. UL37COOH is preferentially ER-localized and extensively N-glycosylated, and the stable
traffics from the ER to mitochondria. Although they share most of their ORFs (aa 1162), their different C termini and, specifically, the second hydrophobic domain at aa 178196 suggest that
is structurally different from pUL37x1. These structural differences may have functional significance for HCMV growth in humans considering their preferential trafficking to mitochondria.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 March 2004;
accepted 8 April 2004.