The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus.

Members of the Rab subfamily of small GTPases play an important role in the regulation of intracellular transport routes. Rab6A has been shown to be a regulator of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER). Here, we report on the identification of a Rab6 isoform, termed Rab6B. The corresponding full-length cDNA was isolated from a Caco-2 cell library. The deduced amino acid sequence showed 91% identity with the Rab6A protein and revealed that sequence divergence is dispersed over a large region of the COOH-terminal domain. Rab6B is encoded by an independent gene which is located on chromosome 3 region q21-q23. In contrast to Rab6A whose expression is ubiquitous, northern blot analysis, immunohistochemistry, and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma cell line SK-N-SH. In brain, Rab6B was found to be specifically expressed in microglia, pericytes and Purkinje cells. Endogenous Rab6B localises to the Golgi apparatus and to ERGIC-53-positive vesicles. Comparable studies between Rab6A and Rab6B revealed distinct biochemical and cellular properties. Rab6B displayed lower GTP-binding activities and in overexpression studies, the protein is distributed over Golgi and ER membranes, whereas Rab6A is more restricted to the Golgi apparatus. Since the GTP-bound form of Rab6B (Rab6B Q72L) does interact with all known Rab6A effectors, including Rabkinesin-6, the results suggest a cell-type specific role for Rab6B in retrograde membrane traffic at the level of the Golgi complex.

Routing and processing of lactase-phlorizin hydrolase in transfected Caco-2 cells.

Human lactase-phlorizin hydrolase (LPH) is a digestive enzyme that is expressed in the small intestinal brush-border membrane. After terminal glycosylation in the Golgi apparatus, the 230-kDa pro-LPH is cleaved into the 160-kDa brush-border LPHbeta and the 100-kDa profragment (LPHalpha). Since LPHbeta is not transport-competent when it is expressed separately from LPHalpha in COS-1 cells, it was suggested that LPHalpha functions as an intramolecular chaperone. What happens to LPHalpha after cleavage is still unclear. To analyze and localize LPHalpha in polarized epithelial cells, wild type and tagged LPH were stably expressed in Caco-2 cells. In tagged LPH, a vesicular stomatitis virus epitope tag was inserted into the LPHalpha region. Wild type and tagged proteins were processed at similar rates, and both cleaved LPHbeta forms were expressed at the apical cell surface. Pro-LPH was recognized by antibodies against LPH, a profragment epitope and the vesicular stomatitis virus tag. LPHalpha alone, however, could not be recovered by these antibodies. Our data suggest that LPHalpha is degraded immediately after cleavage.

The use of backscattered electrons in analytical electron microscopy for the measurement of the mass of individual rat blood platelets.

The backscattered electron signal, generated in individual cells, has been used to measure the dry mass of these cells. Absolute mass values were obtained by comparing the backscattered electron signals of cells to the signals of polystyrene-latex spheres of known mass. The technique was carried out in an automated analytical scanning transmission electron microscope and applied to rat blood platelets. The resulting mass distributions agreed well with the distribution measured with a method that uses the transmitted electron signal by means of densitometric analysis of electrographs. Also the range of masses was in agreement with values deduced from data in the literature. The fully automated technique has the advantage that it is direct, fast, and that thicker specimens can be measured than is possible using the transmitted electron signal. The method is intended for use in combination with quantitative electron probe X-ray microanalysis and is then able to produce elemental mass fractions of biological specimens at the subcellular level.

Quantitative electron probe x-ray microanalysis and densitometric mass determination of individual rat blood platelets.

An improved method for quantitative electron probe X-ray microanalysis of thin biological specimens, introduced recently, has been applied to the elemental analysis of rat blood platelets. The method uses the X-ray signal to quantify the elemental content of an object and electron scattering to determine the total dry mass of the object. Along with the dry mass distribution, data were obtained on the content and mass fraction of calcium, magnesium, and sulfur in 31 individual platelets. The mean platelet dry mass was found to be 985 fg. The mean Ca, Mg, and S contents were 1.80, 3.41, and 18.3 fg, with mean dry mass fractions of 0.195, 0.396, and 1.96%, respectively. Furthermore, these elements appear to be unevenly distributed among the platelet population.

Mass determination of thin biological specimens for use in quantitative electron probe X-ray microanalysis.

In the quantitative electron probe X-ray microanalysis of thin specimens the total mass thickness of a specimen is often required to express the elemental content as fractions of the total mass. In the present paper three methods for the measurement of mass thickness of thin specimens are reviewed and compared as to their applicability for use in quantitative X-ray microanalysis. The methods are based on the use of the continuum X-ray intensity, the backscattered electron intensity, and the transmitted electron intensity, respectively. From experimental results, it is concluded that the mass measurement method, based on the transmitted electron intensity, gives more accurate mass values than does the continuum X-ray method. The use of an independent mass measurement method is advantageous when low temperature oxygen plasma ashing is applied to lower X-ray analysis detection limits. In this way, elements present in ashed specimens can be expressed as fractions of the original specimen mass.