Immunocytochemical localization of the multicatalytic proteinase (Proteasome) in crustacean striated muscles

Abstract

Multicatalytic proteinase (MCP) is thought to play a central role in the processing and turnover of intracellular proteins in eukaryotic cells. Immunocytochemistry was used to determine the intracellular distribution of the MCP in the claw muscles of the land crab, Gecarcinus lateralis, and the claw and abdominal muscles of the American lobster, Homarus americanus. Cryosections were stained with an affinity-purified polyclonal antibody to lobster MCP that cross-reacted with the land crab enzyme. Two types of staining were observed: a diffuse cytoplasmic staining, and a dense aggregate staining primarily associated with invaginations of the cell membrane. The cytoplasmic staining appeared reticulated in favorable transverse sections due to a preferential localization of MCP to the intermyofibrillar space. The aggregate staining was associated with neither nuclei nor mitochondria, since stains specific for these organelles (Hoechst stain and nicotinamide adenine dinucleotide diaphorase histochemistry, respectively) did not colocalize with the aggregates. Trypsinlike peptidase activities of isolated microsomal and postmicrosomal fractions indicated that less than 1% of the total MCP was associated with the microsomal fraction. Immunoprecipitation of the same fractions confirmed the presence of MCP in the microsomes as well as in the cytosol. These results suggest that the MCP is primarily associated with cytoplasmic components; the aggregate staining may result from the association of the MCP with cellular membrane systems.

Proteasomes in Distal Myopathy with Rimmed Vacuoles.

“…Proteasomes are distributed both in the cytoplasm and in the nuclei of skeletal muscle fibers in various species (15,16). In the present immunohistochemical study, their distribution in normal humanmuscleswas similar to that seen in animal muscle, although the intensity was very weak.…”

“…Significant elevation in enzyme activity, protein amount, or protease mRNA levels, as well as elevations of ubiquitinated proteins and ubiquitin, have been observed in those muscles (8, 10, 1 1, 14, 20). To our knowledge, however, localization of proteasomes in normal and diseased humanmuscle has not been studied until now, although some animal studies have been reported (15)(16)(17).…”

A complex between peptide: N -glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins

“…Misfolded proteins targeted for proteolysis via the ERAD pathway would be expected to accumulate around the ER. Previous analysis using electron microscopy determined that 14% of the cytoplasmic proteasome is associated with the ER (27,37), whereas subcellular fractionation methods estimate that only 1% of proteasome is bound to the ER (38). This discrepancy may be due to disruption of the ER-proteasome interaction during subcellular fractionation.…”

Lobster Muscle Proteasome and the Degradation of Myofibrillar Proteins

“…Vertemuscle fibers that mediate excitation-contrac-brate protéasomes generally have higher chytion coupling. These aggregates appear to be motrypsin-like and BrAAP activities, and the origin of the small amount (< 1 %) of pro-lower trypsin-like activity than the lobster teasome found in microsomal fractions [21]. proteasome [5,9,10].…”

“…It is easily extracted from muscle with low-salt buffers and greater than 99% of the activity is found in the post-et al [22], since the nuclei of connective tissue microsomal fraction [21]. Immunocytochem-cells in the same sections are stained with the ical staining shows a diffuse cytoplasmic antibody ( fig.…”