Electron microscopic inspection of the ∆ sfb2/sec24-11 mutant….… 99

By electron microscopic inspection of potassium permanganate fixed cells we tried to look whether there were morphological differences between wild type (MSUC-3D), ∆sfb2 (RPY61), sec24-11 (RPY18) and ∆sfb2/sec24-11 (RPY72) mutants. Cells were grown at 25°C to mid-log phase, then incubated at 37°C for 1h before fixation.

Compared to wild type (Fig. 4.31.A), ∆sfb2 (Fig. 4.31.B) and sec24-11 (Fig. 4.31.C) mutant cells had an about 2-fold increase in the number of 30-50 nm vesicles (indicated by arrowheads). Interestingly, the ∆sfb2/sec24-11 double mutant (Fig.

4.31.D) exhibited enhanced proliferation of ER membranes, and a further increase (approximately 3-fold compared to wild type) of vesicular structures many of which formed aggregates (Fig. 4.31.E); these aggregates were present in about 20 % of the sections. The aggregated structures were of round and sometimes of short rod-like appearance. In some cases it can be seen that they are surrounded by a membrane.

4.2.9 Immunofluorescence detection of one member of the mammalian Sec24p family in monkey CV1 cells

When this work was started, the mammalian homologues of Sec24p were unknown. By database search, a full-length human cDNA clone named KIAA0079 was found sharing significant homology with Sec24p (GenBank accession number D38555, kindly provided to us by Dr. Nomura from the Kazusa DNA research institute, Japan; (Nomura et al., 1994). The cDNA encodes a 1094 amino acid-long protein with a calculated molecular mass of 118 kDa which is 27.2% identical to Sec24p in 960 overlapping amino acids, 27.8% identical to Sfb2p in 802 overlapping amino acids and 26.4% identical to Sfb3p in 904 overlapping amino acids. The sequence present in the database contains a mistake (an extra base at position 3233 and consequently a frame-shift after amino acid 1040). The sequence shown in Fig.

4.32 is the correct one. This sequence mistake was found after multiple alignment with different ESTs and subsequently confirmed by sequencing.

In order to produce antibodies against this protein, different fragments were cloned and expressed as 6xHis fusion proteins. The two protein fragments, highlighted in Fig. 4.32, corresponding to the sequence regions 365-522 (for simplicity named peptide 2) and 747-992 (peptide 5), respectively, were used as antigens.

M M

M

V

V

V

V N

N

N

N

ER A

B

C

D

E

Fig. 4.31 Thin-section electron-micrographs of wildtype (MSUC-3D; A), ∆sfb2 (RPY61; B), sec24-11 (RPY18; C) and ∆sfb2/sec24-11 (RPY72; D) mutants. Cells were grown at 37°C for 1 h before potassium permanganate fixation. (E) enlarged view of ∆sfb2/sec24-11 double mutant cells showing clusters of round and rod-like membrane-bounded structures. Vesicle aggregates are indicated by an arrow in D, and 30-50 nm vesicles by arrowheads. N= nucleus, V= vacuole, M= mitochondria.

1 µm

0.1 µm

The two 6xHis-tagged fragments were expressed in bacteria containing the expression plasmids pQE30-KIAA0079(363-522), pQE50-KIAA0079(363-522) and pQE30-KIAA0079(747-992) (see Table 7.6). The methods used to produce and purify these antibodies are described in Section 3.5. In Fig. 4.33 an immunoblot of protein extracts from Hela and CV1 cells using the antibodies anti-KIAA0079-2 (serum from rabbits 166 and 167) and anti-KIAA0079-5 (serum from rabbits 168 and 169) is shown.

1 MNVNQSVPPV PPFGQPQPIY PGYHQSSYGG QSGSTAPAIP YGAYNGPVPG 51 YQQTPPQGMS RAPPSSGAPP ASTAQAPCGQ AAYGQFGQGD VQNGPSSTVQ 101 MQRLPGSQPF GSPLAPVGNQ PPVLQPYGPP PTSAQVATQL SGMQISGAVA 151 PAPPSSGLGF GPPTSLASAS GSFPNSGLYG SYPQGQAPPL SQAQGHPGIQ 201 TPQRSAPSQA SSFTPPASGG PRLPSMTGPL LPGQSFGGPS VSQPNHVSSP 251 PQALPPGTQM TGPLGPLPPM HSPQQPGYQP QQNGSFGPAR GPQSNYGGPY 301 PAAPTFGSQP GPPQPLPPKR LDPDAIPSPI QVIEDDRNNR GTEPFVTGVR 351 GQVPPLVTTN FLVKDQGNAS PRYIRCTSYN IPCTSDMAKQ AQVPLAAVIK 401 PLARLPPEEA SPYVVDHGES GPLRCNRCKA YMCPFMQFIE GGRRFQCCFC 451 SCINDVPPQY FQHLDHTGKR VDAYDRPELS LGSYEFLATV DYCKNNKFPS 501 PPAFIFMIDV SYNAIRTGLV RLLCEELKSL LDFLPREGGA EESAIRVGFV 551 TYNKVLHFYN VKSSLAQPQM MVVSDVADMF VPLLDGFLVN VNESRAVITS 601 LLDQIPEMFA DTRETETVFV PVIQAGMEAL KAAECAGKLF LFHTSLPIAE 651 APGKLKNRDD RKLINTDKEK TLFQPQTGAY QTLAKECVAQ GCCVDLFLFP 701 NQYVDVATLS VVPQLTGGSV YKYASFQVEN DQERFLSDLR RDVQKVVGFD 751 AVMRVRTSTG IRAVDFFGAF YMSNTTDVEL AGLDGDKTVT VEFKHDDRLN 801 EESGALLQCA LLYTSCAGQR RLRIHNLALN CCTQLADLYR NCETDTLINY 851 MAKFAYRGVL NSPVKAVRDT LITQCAQILA CYRKNCASPS SAGQLILPEC 901 MKLLPVYLNC VLKSDVLQPG AEVTTDDRAY VRQLVTSMDV TETNVFFYPR 951 LLPLTKSPVE STTEPPAVRA SEERLSNGDI YLLENGLNLF LWVGASVQQG 1001 VVQSLFSVSS FSQITSGLSV LPVLDNPLSK KVRGLIDSLR AQRSRYMKLT 1051 VVKQEDKMEM LFKHFLVEDK SLSGGASYVD FLCHMHKEIR QLLS

Fig. 4.32 Protein encoded by the cDNA KIAA0079 (hSec24Cp). The sequences highlighted represent the two fragments (for simplicitynamed peptides 2 and 5) used to produce anti-KIAA0079-2 and anti-KIAA0079-5 antibodies, respectively. The putative zinc finger domain is underlined.

Fig. 4.33 Protein extracts from Hela (H) and CV1 (C) cells were separated by SDS-PAGE and immunoblotted with serum from two rabbits (166, 167) immunized with "peptide 2" and from two rabbits (168, 169) immunized with "peptide 5".

The calculated molecular mass of the KIAA0079-encoded protein is 118 kDa.

H C H C H C H C kDa

120

-

87-166 167 168 169

By indirect immunofluorescence with anti-KIAA0079 antibodies it was possible to judge the subcellular localization of KIAA0079-encoded protein in methanol-acetone fixed CV1 cells (see Methods 3.6.3). The following proteins were immuno-labeled and used for reference: protein disulfide-isomerase (PDI), β1 and β2 adaptins and Golgi 58K. PDI is a soluble ER resident protein containing at its C-terminus the highly conserved KDEL sequence (Kaetzel et al., 1987); β1 and β2 adaptins are components of the adaptor complexes AP-1 and AP-2, the antibodies against β1 and β2 adaptins

anti-KIAA0079-2 (166)

anti-β1 and β2

adaptins

Merge ^{10 µm}

anti-KIAA0079-2 (166)

anti-Golgi K58 Merge ^{10 µm}

anti-KIAA0079-2 (167)

anti-PDI Merge ^{10 µm}

Fig. 4.34 Double immunofluorescence of methanol-acetone fixed CV1 cells using anti-KIAA0079-2 antibody in combination with the antibodies indicated in the panels. The secondary antibodies were rhodamine red-X-conjugated and Oregon-Green-488-conjugated.

Confocal images were taken using a Leica TCS NT confocal laser scanning microscope.

stain clathrin-coated domains at the plasma membrane and at the Golgi region (Ahle et al., 1988); Golgi 58K is a microtubule-binding peripheral Golgi membrane protein (Bloom and Brashear, 1989).

As can be seen in Fig. 4.34, the anti-KIAA0079 antibodies revealed a punctate pattern scattered throughout the cytoplasm along with some concentration at the perinuclear region (overlapping with Golgi markers). Some of the punctate structures were overlapping with ER structures, as can be seen in the double staining with PDI.

This distribution pattern is similar to what was previously described for another COPII component, the mammalian Sec13p (Tang et al., 1997). The same was subsequently observed by other research groups (Pagano et al., 1999; Tang et al., 1999; Tani et al., 1999), who described the entire family of mammalian Sec24 proteins: hSec24Ap, hSec24Bp, hSec24Cp, hSec24Dp. The KIAA0079-encoded protein is hSec24C.

hSec24Ap and hSec24Bp seem to form one class sharing about 56% identity, while hSec24Cp and hSec24Dp form another class with about 52% identity. There is about 20% identity between the two pairs (Tang et al., 1999), which is almost the same degree of identity observed for Sec24p, Sfb2p (56% identity between them) and Sfb3p (23% identity with Sec24p and 24% with Sfb2p).