Tài liệu Báo cáo khoa học: Hu-K4 is a ubiquitously expressed type 2 transmembrane protein associated

Hu-K4 is a ubiquitously expressed type 2 transmembrane

protein associated with the endoplasmic reticulum

Antonia Munck, Christopher Bo¨ hm, Nicole M. Seibel, Zara Hashemol Hosseini and

Wolfgang Hampe

Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology II: University Hospital Eppendorf, Hamburg, Germany

The Hu-K4 protein was first identified as a human

homologue of the K4L protein of vaccinia virus [1].

K4L is a nonessential protein in the life cycle of the virus

and has unknown function. Both Hu-K4 and K4L con￾tain two HXKXXXXD ⁄E (HKD) motifs which make

them members of the superfamily of HKD proteins

together with phospholipase D proteins and phospholi￾pid synthases [2]. The closest homologues of Hu-K4 are

found in other mammals, murine SAM9 [3] has 93%

identical amino acid residues (Fig. 1). More distantly

related proteins are found in Xenopus (54%) and Dro￾sophila (48%) and in vaccinia virus. In addition to the

viral K4L protein (48%) this virus also encodes the clo￾sest relative of Hu-K4 with known function, the most

abundant viral protein p37 (21%). Other members of

the HKD superfamily are the phospholipase D iso￾forms. Like the other proteins shown in Fig. 1 they

harbour two HKD motifs which are involved in the

catalytic process [4]. For this reason Hu-K4 was named

phospholipase D3 in the GenBank entry NP_036400

although outside the HKD motifs no similarity exists.

Phospholipase D enzymes catalyse the hydrolysis of

membrane phospholipids, e.g. of phosphatidyl choline

to choline and phosphatidic acid which was ascribed

a second-messenger function. Two isoforms, phospho￾lipase D1 and D2, are well characterized and part of

different signalling cascades implicated in membrane

trafficking, cytoskeletal reorganization, receptor endo￾cytosis, exocytosis, cell migration, and regulation of the

cell cycle [5]. For the murine orthologue of Hu-K4,

SAM9, so far no phospholipase D activity could be

assigned indicating that Hu-K4 and SAM9 might have

another function [3].

The above mentioned protein p37 is essential for

efficient cell-to-cell spreading by vaccinia virus [6].

During maturation of the virus, p37 is required for the

Keywords

topology, subcellular localization, gene

structure, expression pattern, translational

control

Correspondence

W. Hampe, Institut fu¨r Biochemie und

Molekularbiologie II, Molekulare Zellbiologie,

Universita¨tsklinikum Eppendorf, Martinistr.

52, D-20246 Hamburg, Germany

Fax: +49 40 42803 4592

Tel: +49 40 42803 9967

E-mail: [email protected]

(Received 2 December 2004, revised 1

February 2005, accepted 8 February 2005)

doi:10.1111/j.1742-4658.2005.04601.x

Hu-K4 is a human protein homologous to the K4L protein of vaccinia

virus. Due to the presence of two HKD motifs, Hu-K4 was assigned to the

family of Phospholipase D proteins although so far no catalytic activity

has been shown. The Hu-K4 mRNA is found in many human organs with

highest expression levels in the central nervous system. We extended the

ORF of Hu-K4 to the 5¢ direction. As a consequence the protein is 53

amino acids larger than originally predicted, now harbouring a putative

transmembrane domain. The exon ⁄ intron structure of the Hu-K4 gene

reveals extensive alternative splicing in the 5¢ untranslated region. Due to

the absence of G ⁄ C-rich regions and upstream ATG codons, the mRNA

isoform in brain may be translated with higher efficacy leading to a high

Hu-K4 protein concentration in this tissue. Using a specific antiserum pro￾duced against Hu-K4 we found that Hu-K4 is a membrane-bound protein

colocalizing with protein disulfide isomerase, a marker of the endoplasmic

reticulum. Glycosylation of Hu-K4 as shown by treatment with peptide

N-glycosidase F or tunicamycin indicates that Hu-K4 has a type 2 trans￾membrane topology.

Abbreviations

EST, expressed sequence tag; GST, glutathione S-transferase; Hu-K4, human K4L homologue; PNGaseF, peptide N-glycosidase F.

1718 FEBS Journal 272 (2005) 1718–1726 ª 2005 FEBS